High-Potency Botulinum Toxin Formulations

ABSTRACT

The present invention provides improved formulations of botulinum toxin that increase delivery of the botulinum toxin to neural and associated tissues and exhibit a higher specific neurotoxicity and higher potency (in LD 50  Units) than available formulations of botulinum toxins. These improved formulations enable physicians to treat a wide variety of pathological conditions with a lower toxin load that reduces the risk of inducing an immune response against the toxin and its associated proteins that may ultimately lead to the development of toxin resistance. These benefits are particularly important in the treatment of conditions that require high-dose or chronic administration of botulinum toxin. Additionally, the decreased in LD 50  Unit doses of inventive formulations allows for controlled administration limits diffusion. The present invention also provides methods of treating neuromuscular diseases and pain, using low-dose botulinum toxin.

This application is a continuation of U.S. patent application Ser. No.12/662,186, filed Apr. 5, 2010, which is a continuation of U.S. patentapplication Ser. No. 11/111,951, filed Apr. 22, 2005, and issued as U.S.Pat. No. 7,691,394, which is a continuation-in-part of both (1) U.S.patent application Ser. No. 10/740,755, filed Dec. 22, 2003, and issuedas U.S. Pat. No. 7,491,403, which claims benefit to U.S. ProvisionalApplication Ser. No. 60/435,901, filed on Dec. 20, 2002, and (2) U.S.patent application Ser. No. 10/446,562, filed on May 28, 2003, andissued as U.S. Pat. No. 7,459,164, which claims benefit to U.S.Provisional Application Ser. No. 60/383,570, filed May 28, 2002, all ofwhich are hereby incorporated by reference herein in their entirety.

TECHNICAL FIELD OF THE INVENTION

This invention relates to pharmaceutical formulations of botulinumneurotoxin that have high specific neurotoxicity and that allow thetherapeutically effective use of fewer LD₅₀ Units in clinicalapplications. The invention further relates to methods for the treatmentof a variety of neuromuscular diseases, pain, inflammatory and cutaneousdisorders with low-dose neurotoxin formulations based both on thedelivery of reduced mass of neurotoxin and lower LD₅₀ Units.

BACKGROUND OF THE INVENTION A. Botulinum Toxin: Mechanism of Action

Botulinum neurotoxin is a toxin isolated from a strain of Clostridiumbotulinum, that acts at the neuromuscular junction by inhibiting releaseof acetylcholine. Botulinum toxin is initially formed as a single-chainpolypeptide that is cleaved to form a light chain that is bound to aheavy chain through a disulfide bond. The denervating effect ofbotulinum toxin occurs through: 1) the binding of the heavy chain tohigh-affinity receptors at the presynaptic terminal; 2) internalizationof botulinum toxin through endocytosis; 3) translocation of the lightchain into the cytoplasm of the nerve terminal; and 4) theendo-metalloprotease activity of the light chain (zinc cofactor) cleavesspecific synaptic proteins that inhibit fusion of synaptic vesicles withthe presynaptic membrane, thereby inhibiting the release ofacetylcholine contained in the vesicles. Absent acetylcholine, themuscle does not receive the necessary signal for the muscle to contract.Subsequent to injection, neurogenic muscular atrophy ensues afterseveral weeks.

B. Botulinum Toxin: Clinical Applications

A deadly toxin at high concentrations and quantities, botulinum toxinhas been used as a valuable therapeutic for the treatment of manyneuromuscular diseases (e.g., dystonia, hemifacial spasm, bruxism,spasticity, cerebral palsy, torticollis), as well as sensory disordersand cutaneous disorders (myofascial pain, migraine, tension headaches,neuropathy, hyperhydrosis), and in the treatment of disorders involvinginflammation. The therapeutic value of botulinum toxin in its ability toproduce local regional denervation of specific muscles and tissues.

The action of botulinum toxin on nerve terminals is irreversible. Axonsprouting, however, reverses the denervating effects of the toxin withintwo to six months. Consequently, a variety of conditions and disordersrequire repeated administration of the neurotoxin. Resistance tobotulinum toxin is an important clinical consequence and problemresulting from repeated administration of botulinum toxin and theproduction of neutralizing antibodies. (Naumann et al. (1998) J. Neurol.Neurosurg. Psychiatry 65: 924-927; Hauna et al. (1998) J. Neurol.Neurosurg. Psychiatry 66: 612-616). The problem is most noted inhigh-dose applications such as cervical dystonia, however, immunity andresistance to the botulinum neurotoxin may occur with lower doseapplications such as blepharospasm. Recently, the inventor observed thatresistance can occur even with low-dose cosmetic applications, such asthe treatment of facial rhytides. Accordingly, it is an object of thepresent invention to provide high-potency formulations and correspondingmethods that reduce the likelihood of neutralizing antibodies insubjects treated with botulinum toxin.

The antigenicity of botulinum toxin stimulates antibody formation thatreduces and most often completely obliterates the therapeuticeffectiveness of botulinum-neurotoxin-based pharmaceuticals and mayultimately lead to abandonment of botulinum therapy. Several strategiesto minimize the development of resistance have been directed towardreducing the antigenicity of the botulinum neurotoxin itself. Forexample, pegylated botulinum toxin (botulinum toxin covalently coupledto polyethylene glycol) have been developed for the treatment ofneuromuscular disorders. Pegylation of the toxin is site directedthereby reducing antigenicity without interfering with neurotoxiceffect. (See, U.S. Patent Publication No. 20020197278). Also,hybrid-toxin molecules with reduced antigenicity have been synthesizedusing the targeting and internalization portion (heavy chain) of onetoxin serotype and the catalytic portion of a different serotype (lightchain). The hybrid-toxin molecules exhibit reduced antigenicity butretain the inherent-binding specificity of the botulinal-heavy chainfrom the first serotype and the catalytic potency of the light chainfrom the second serotype. (See, U.S. Pat. No. 6,444,209).

Reduced antigenicity may also be achieved by further purifying theneurotoxin by reducing the antigenic complex proteins and otherclostridial proteins associated with the toxin. (See, U.S. Pat. Nos.5,756,468 and 5,512,547). Type A neurotoxin produced by C. botulinum ispresent as part of a complex of at least seven different noncovalentlybound proteins. These nontoxic proteins range in size from about 17 to118 kD and are associated with the neurotoxin that has a molecularweight of about 147 kD. (Goodnough et al. (1993) Appl. Environ.Microbiol. 59: 2339-2342; Gimenez et al. (1993) Protein Chem. 12:349-361; DasGupta (1980) Canad. J. Microbiol. 26: 992-997). Some of thenon-toxic proteins associated with the various toxin complexes havehemagglutinating abilities (Sugiyama (1980) Microbiol. Rev. 44: 419-448;Somers et al. (1991) J. Protein Chem. 10:415-425). In particular,non-neurotoxic fractions of the L complexes of type A, B, C, and D havebeen shown to have hemagglutinating activity. Hemagglutinin fractionsisolated from the different serotypes show some serologicalcross-reactivity. Non-toxic fractions from type A and B serotypescross-react (Goodnough and Johnson (1993) Appl. Environ. Microbiol. 59:2339-2342) as do non-toxic fractions from types E and F. The non-toxicfractions of types C₁ and D are antigenically identical as determined byOuchterlony diffusion (Sakaguchi et al. (1974) Jpn. J. Med. Sci. Biol.27: 161-170). By removing these proteins, more neurotoxin may bedelivered to a therapeutic site with less antigenic proteins that maylead to the production of neutralizing antibodies.

C. Complications Associated with Conventional Botulinum-ToxinFormulations.

Substantial differences in the complication rate have also been noted attherapeutic quantities of different botulinum preparations. Side effectssuch as those resulting from diffusion of the botulinum toxin from thesite of administration appear to be dependent on the formulation ofbotulinum toxin. For instance, dysphagia rates (difficulty swallowing)is a well-known complication of botulinum toxin administration when usedfor the treatment of cervical dystonia. (Borodic et al. (1990) BotulinumA toxin for the treatment of spasmodic torticollis. Dysphagia andRegional Toxin Spread. Head & Neck, 12: 392-398; incorporated herein byreference in its entirety). Differences in the rate of this complicationbetween various formulations has been well appreciated when reviewingprior art literature between 1984-1995. Furthermore differences in therate of ptosis (drooping eyelid) have been reported when comparingvarious immunotypes and different preparations of the same immunotype(see Table 1). It has become well accepted that this complication is theresult of diffusion of botulinum toxin away from the injections sites, aproperty which is in conflict with the clinical goal of containing thedenervating or biologic effect to a specific target region. Theformulations and methods disclosed herein contain the biologic effect ofthe neurotoxin to a targeted anatomic region and thereby reduce thediffusion potential of the botulinum toxin pharmaceutical and decreasethe associated side effects.

TABLE 2 Diffusion-related complications between various pharmaceuticalformulations of botulinum toxin. Complication BOTOX ® DYSPORT ®²MYOBLOC ®³ Ptosis¹ <2% 12-15% 30-40% Dysphagia <2% 14-21% 10-17%¹Nussgens et al. (1997) Comparison of two botulinum-toxin preparationsin the treatment of essential blepharospasm. Graefes Arch Clin ExpOphthalmol 235(4): 197-199. ²Phase 3 Studies 1998-1989 for OculinumMeta-analysis of clinical studies on Dysphagia and Botulinum 1995 at NIH(Borodic). ³Lew et al. (1997) Botulinum toxin type B: a double-blind,placebo-controlled, safety and efficacy study in cervical dystonia.Neurology 49(3): 701-707.

In 1991, Borodic et al. developed a histologic model demonstrating ahistochemical and morphologic diffusion gradient from point injectionsof botulinum toxin. (Borodic et al. (1991) Botulinum toxin: Clinical andscientific aspects. Opthamology Clinics of North America 4: 491-503;incorporated herein by reference in its entirety). The gradient was dosedependent over single muscle strips and capable of crossing fascialplanes. The diffusion model was further demonstrated on the facialwrinkling pattern of the human forehead. (Borodic et al (1992) Botulinumtoxin for spasmodic torticollis, multiple vs single point injections permuscle. Head and Neck 14: 33-37). Diffusion was thereafter used toexplain the mechanism for dysphagia after surface injections ofbotulinum injection for the human neck and ptosis (drooping eyelid)after periocular injections for the treatment of essentialblepharospasm. Ptosis results from diffusion of neuromuscular blockingactivity from the lid edge to the muscular portion of the upper eyelidretractor, which lies in the upper orbital space. Dysphagia results fromdiffusion of neuromuscular weakening effect from the sternomastoidmuscle, targeted for treatment of torticollis, to the peripharygealmusculature which generates the force for effective swallowing. Fromboth histologic models and clinical experience, diffusion appearsdirectly related to the quantity of toxin (in LD₅₀ units) administered.Consequently, the greater the quantity of toxin used as an injection inunits used, the greater the diffusion from that point. A review of thescientific literature from the 1980's and early 1990's reveals thatdysphagia is more commonly with observed with DYSPORT® than BOTOX®.Recently, from studies done at European centers, the differences indysphagia rates have been confirmed (Ranoux et al. (2002) Respectivepotencies of DYSPORT® and BOTOX®: a double blind, randomized, crossoverstudy in cervical dystonia. J. Neurol. Neurosurg. Psychiatry 72:459-462). Differences in ptosis rates for the treatment of blepharospasmhave also been observed comparing BOTOX®. Ptosis is less frequentlyobserved with BOTOX® (Nussgens et al. (1997) Comparison of twobotulinum-toxin preparations in the treatment of essentialBlepharospasm. Graefes Arch Clin Exp Ophthalmol. 235(4): 197-199). Majordifferences in the ptosis complication have also been reported whenusing botulinum toxin type B for the treatment of glabellar and foreheadwrinkles when compared to botulinum type A (BOTOX®). (Holck et al.Comparison of High Dose Botulinum Toxin Type B to Botulinum Type A inthe Treatment of Lateral Canthal Rhytides American Society of OphthalmicPlastic and reconstructive Surgeons Annual Meeting, Anaheim, Calif.11-14-03).

Prior to this invention, the in vivo binding of sequestration agents,such as albumin, to botulinum toxin has never been identified asimportant to clinical effectiveness of botulinum-toxin-basedpharmaceuticals. By enhancing regional sequestration of the neurotoxinand facilitating saturation of neurotoxin receptors on neural tissues,high-concentration-albumin formulations improve the clinicaleffectiveness of botulinum toxin and reduce side effects such as thoseresulting from diffusion of the botulinum toxin from the site ofadministration. There has been no prior suggestion that increasing thealbumin concentration, for example, relative to the neurotoxin, couldenhance the effectiveness for the treatment of human disease. Theexisting botulinum toxin preparations currently available for clinicalpractice are BOTOX®, DYSPORT®, and MYOBLOC®. The present inventionidentifies the mechanism and provides compositions of improved utilityof botulinum-toxin-based pharmaceuticals by increasing the concentrationof a sequestration agent and other viscous agents to enhancesequestration and improve the effectiveness where other availablebotulinum toxin preparations have failed.

D. Sequestration.

Albumin was initially used to formulate botulinum-toxin-basedpharmaceuticals because of its stabilizing effect on the biologicactivity of the neurotoxin at high dilutions (see Schantz, BotulinumToxin Therapy, Marcel Dekker 1994). Dilution of the purified botulinumtoxin crystals with physiologic saline or water would cause the biologicactivity and pharmaceutical properties to be lost at high dilutions.Additionally, the albumin has been reported to help keep the neurotoxinmolecule from binding to glass containers. During the pre-clinicaldevelopment of BOTOX® or any other botulinum toxin prepared forpharmaceutical use, there was no appreciation for the importance ofalbumin in the formulation other than a dilution stabilizer andexcipient to keep the neurotoxin from binding to glass.

BOTOX® and DYSPORT® are derived from different strains of Clostridialspecies. BOTOX® is derived from the Hall strain of Clostridium botulinumoriginally maintained by the University of Wisconsin, whereas DYSPORT®is derived from British Microbiology Collection. Immunologic crossreactivity exists between the products as both products were derivedfrom immunotype A strains. Despite similar immunotypes, the clinicalresponses between BOTOX® and DYSPORT® may be explained by thedifferences in the excipients used in each formulation. The differencein human serum albumin concentrations between BOTOX® and DYSPORT® areoutlined in Table 3.

TABLE 3 Human Serum Albumin content of various pharmaceuticalformulations of botulinum toxin. Formulation Albumin¹ LD₅₀/μg albuminBOTOX ® 500 μg 0.2 DYSPORT ® 125 μg 5.0 ¹Albumin is represented in mgper 100 LD₅₀ units of botulinum toxin. Other differences exist includingthe presence of stabilizing sugars, Lactose is used in DYSPORT ® and notused in BOTOX ®.

The albumin discrepancy between BOTOX® and DYSPORT® is almost identicalto the difference in dose requirements observed between BOTOX® andDYSPORT® in multiple clinical studies. The correlation between thealbumin ratio/clinical potency ratio is further strengthened by changesin pharmacologic properties of DYSPORT® when albumin is added to thevials using a mouse hemidiaphram animal model. Wohlfahrt et al. notedusing this model that adding albumin to one vials of DYSPORT® broughtbiologic activity higher using the mouse hemi-diaphragm model. (Biglalkeet al (2001) Botulinum A toxin: DYSPORT® improvement of biologicalavailability. Exp. Neurol. 168(1): 162-170). The authors suggested theincreased biologic activity resulted from increased stability asmeasured with the mouse LD₅₀ bioassay afforded by the albuminconcentration increase. (Biglalke et al (2001) Botulinum A toxin:DYSPORT® improvement of biological availability. Exp. Neurol. 168(1):162-170). The authors explained the differences of albumin on the LD₅₀bioassay without reference to mechanism of action in tissues orpharmacologic-pharmacokinetic importance, that is, in vivo albuminbinding, enhanced sequestration, and improvement in therapeutic effects.The same authors further observed in a rat-diaphragm preparation, thatthe addition of albumin to the BOTOX® preparation could notsubstantially increase regional denervative effects and did not advocateany changes in formulation. The findings of these researchers concludedthat there was an effect of the albumin concentration on the LD₅₀measurements, however, their work did not demonstrate any increasedpotency of BOTOX® on regional denervation or that DYSPORT® could beenhanced to give any greater denervation potency over BOTOX®. Their workwas limited by the in vitro nature of their experiments, that is, usinga non-blood-perfused-animal dissection of a motor nerve (phrenic nerve)and diaphragm muscle, which fails to accounts for dilutions and tissuefluid flow capable of washing injected toxin away from targeted tissueprior to binding with the nerve axon terminal receptors. The real timeapplication requires an in vivo analysis of the effects of albumin onregional denervation as outlined in the following experiments. Theirwork did identify reasons for differences in LD₅₀ as measured by themouse lethality assay. These workers, however, concluded that noimprovements in potency or effectiveness could be made over existingBOTOX® preparation. (Hanover Germany International Botulinum ToxinMeeting 2002).

Differences in potency, issues relating diffusion and containment of thebiologic effect, and the development of resistance are important in thepharmacology of botulinum-based pharmaceuticals. Described herein is amethod for altering compositions of botulinum based pharmaceuticals toenhance potency, increase sequestration of the botulinum toxin and limitadverse effects of botulinum-based pharmaceuticals.

SUMMARY OF THE INVENTION

The present invention provides a pharmaceutical formulation comprising abotulinum neurotoxin and a sequestration agent present in an amountgreater than about 500 micrograms per 100 LD₅₀ Units neurotoxin, whereinfewer LD₅₀ Units of said formulation are required to achieve atherapeutic response than a formulation comprising botulinum toxin and asequestration agent present in an amount greater than about 500micrograms per 100 LD₅₀ Units neurotoxin. In one embodiment, fewer LD₅₀Units of the pharmaceutical formulation are required to achieve areduction of glabellar lines than a formulation comprising botulinumtoxin and a sequestration agent present in an amount greater than about500 micrograms per 100 LD₅₀ Units neurotoxin. Although the reduction ofglabellar lines may be assessed by any means accepted in the art,patient self-grading and assessment and physician-based photo-scalegrading are preferred. In another embodiment, fewer LD₅₀ Units of thepharmaceutical formulation are required to reduce muscle contraction incervical dystonia than a formulation comprising botulinum toxin and asequestration agent present in an amount greater than about 500micrograms per 100 LD₅₀ Units neurotoxin.

The pharmaceutical formulations of the present invention are comprisedof botulinum toxin and a sequestration agent in an amount greater thanabout 500 micrograms per 100 LD₅₀ Units neurotoxin, wherein thebotulinum toxin may be selected from any one or a combination of thevarious botulinum toxin immunotypes such as A, B, C₁, C₂, C₃, D, E, Fand G. In a preferred embodiment, the botulinum neurotoxin is botulinumtoxin type A. In a preferred embodiment, the sequestration agent ishuman serum albumin or hyaluronate. In a more preferred embodiment, thesequestration agent is human serum albumin. The pharmaceuticalformulations of the present invention may further comprise astabilization or stabilizing agent that stabilizes the activity of thebotulinum neurotoxin. As used herein, “stabilization agent” or“stabilizing agent” means any agent that prolongs the biologic activity,or specifically the neurotoxicity of the botulinum neurotoxin, uponstorage. In a preferred embodiment, the stabilization or stabilizingagent is a monosaccharide or disaccharide. In a more preferredembodiment, trehalose is the stabilization or stabilizing agent.

The pharmaceutical formulations of the present invention are comprisedof botulinum toxin and a sequestration agent in an amount greater thanabout 500 micrograms per 100 LD₅₀ Units neurotoxin. Preferably, thesequestration agent is present in an amount greater than about 500 μgper 100 LD₅₀ Units neurotoxin, 550 μg per 100 LD₅₀ Units neurotoxin, 600μg per 100 LD₅₀ Units neurotoxin, 650 μg per 100 LD₅₀ Units neurotoxin,700 μg per 100 LD₅₀ Units neurotoxin, 750 μg per 100 LD₅₀ Unitsneurotoxin, 800 μg per 100 LD₅₀ Units neurotoxin, 850 μg per 100 LD₅₀Units neurotoxin, 900 μg per 100 LD₅₀ Units neurotoxin, 950 μg per 100LD₅₀ Units neurotoxin, 1000 μg per 100 LD₅₀ Units neurotoxin, 1100 μgper 100 LD₅₀ Units neurotoxin, 1200 μg per 100 LD₅₀ Units neurotoxin,1300 μg per 100 LD₅₀ Units neurotoxin, 1400 μg per 100 LD₅₀ Unitsneurotoxin, 1500 μg per 100 LD₅₀ Units neurotoxin, 1600 μg per 100 LD₅₀Units neurotoxin, 1700 μg per 100 LD₅₀ Units neurotoxin, 1800 μg per 100LD₅₀ Units neurotoxin, 1900 μg per 100 LD₅₀ Units neurotoxin, 2000 μgper 100 LD₅₀ Units neurotoxin, 2250 μg per 100 LD₅₀ Units neurotoxin,2500 μg per 100 LD₅₀ Units neurotoxin, 2750 μg per 100 LD₅₀ Unitsneurotoxin, 3000 μg per 100 LD₅₀ Units neurotoxin, 3250 μg per 100 LD₅₀Units neurotoxin, 3500 μg per 100 LD₅₀ Units neurotoxin, 3750 μg per 100LD₅₀ Units neurotoxin, 4000 μg per 100 LD₅₀ Units neurotoxin, 4250 μgper 100 LD₅₀ Units neurotoxin, 5000 μg per 100 LD₅₀ Units neurotoxin,5250 μg per 100 LD₅₀ Units neurotoxin, 5500 μg per 100 LD₅₀ Unitsneurotoxin, 5750 μg per 100 LD₅₀ Units neurotoxin, 6000 μg per 100 LD₅₀Units neurotoxin, 7000 μg per 100 LD₅₀ Units neurotoxin, 8000 μg per 100LD₅₀ Units neurotoxin, 9000 μg per 100 LD₅₀ Units neurotoxin, or 10,000μg per 100 LD₅₀ Units neurotoxin.

More preferably, the sequestration agent is present in an amount betweenabout 500 and 750 μg per 100 LD₅₀ Units neurotoxin, about 750 and 1000μg per 100 LD₅₀ Units neurotoxin, about 1000 and 1250 μg per 100 LD₅₀Units neurotoxin, about 1250 and 1500 μg per 100 LD₅₀ Units neurotoxin,about 1500 and 1750 μg per 100 LD₅₀ Units neurotoxin, about 1750 and2000 μg per 100 LD₅₀ Units neurotoxin, about 2000 and 2250 μg per 100LD₅₀ Units neurotoxin, about 2250 and 2500 μg per 100 LD₅₀ Unitsneurotoxin, about 2500 and 2750 μg per 100 LD₅₀ Units neurotoxin, about2750 and 3000 μg per 100 LD₅₀ Units neurotoxin, about 3000 and 3250 μgper 100 LD₅₀ Units neurotoxin, about 3250 and 3500 μg per 100 LD₅₀ Unitsneurotoxin, about 3500 and 3750 μg per 100 LD₅₀ Units neurotoxin, about3750 and 4000 μg per 100 LD₅₀ Units neurotoxin, about 4000 and 4250 μgper 100 LD₅₀ Units neurotoxin, about 4250 and 4500 μg per 100 LD₅₀ Unitsneurotoxin, about 4500 and 4750 μg per 100 LD₅₀ Units neurotoxin, about4750 and 5000 μg per 100 LD₅₀ Units neurotoxin, about 5000 and 5250 μgper 100 LD₅₀ Units neurotoxin, about 5250 and 5500 μg per 100 LD₅₀ Unitsneurotoxin, about 5500 and 5750 μg per 100 LD₅₀ Units neurotoxin, about5750 and 6000 μg per 100 LD₅₀ Units neurotoxin, about 6000 and 6250 μgper 100 LD₅₀ Units neurotoxin, about 6250 and 6500 μg per 100 LD₅₀ Unitsneurotoxin, about 6500 and 6750 μg per 100 LD₅₀ Units neurotoxin, about6750 and 7000 μg per 100 LD₅₀ Units neurotoxin, about 7000 and 7500 μgper 100 LD₅₀ Units neurotoxin, about 7500 and 7750 μg per 100 LD₅₀ Unitsneurotoxin, about 7750 and 8000 μg per 100 LD₅₀ Units neurotoxin, about8000 and 8250 μg per 100 LD₅₀ Units neurotoxin, about 8250 and 8500 μgper 100 LD₅₀ Units neurotoxin, about 8500 and 8750 μg per 100 LD₅₀ Unitsneurotoxin, about 8750 and 9000 μg per 100 LD₅₀ Units neurotoxin, about9000 and 9250 μg per 100 LD₅₀ Units neurotoxin, about 9250 and 9500 μgper 100 LD₅₀ Units neurotoxin, about 9500 and 9750 μg per 100 LD₅₀ Unitsneurotoxin, or about 9750 and 10,000 μg per 100 LD₅₀ Units neurotoxin.

The pharmaceutical formulations of the present invention are comprisedof botulinum toxin and a sequestration agent in an amount greater thanabout 500 micrograms per 100 LD₅₀ Units neurotoxin, wherein thebotulinum toxin may be of any purity, as described by specific activityor specific neurotoxicity. In a preferred embodiment, the botulinumtoxin has a specific neurotoxicity of between about 20 and 250 Units/ngneurotoxin, about 50 and 250 Units/ng neurotoxin, about 80 and 250Units/ng neurotoxin, about 90 and 250 Units/ng neurotoxin, about 100 and250 Units/ng neurotoxin, about 150 and 250 Units/ng neurotoxin, or about200 and 250 Units/ng neurotoxin. In a more preferred embodiment, thebotulinum toxin has a specific neurotoxicity of about 20 Units/ngneurotoxin, 30 Units/ng neurotoxin, 40 Units/ng neurotoxin, 50 Units/ngneurotoxin, 60 Units/ng neurotoxin, 70 Units/ng neurotoxin, 80 Units/ngneurotoxin, 90 Units/ng neurotoxin, 100 Units/ng neurotoxin, 110Units/ng neurotoxin, 120 Units/ng neurotoxin, 130 Units/ng neurotoxin,140 Units/ng neurotoxin, 150 Units/ng neurotoxin, 160 Units/ngneurotoxin, 170 Units/ng neurotoxin, 180 Units/ng neurotoxin, 190Units/ng neurotoxin, 200 Units/ng neurotoxin, 210 Units/ng neurotoxin,220 Units/ng neurotoxin, 230 Units/ng neurotoxin, 240 Units/ngneurotoxin, or 250 Units/ng neurotoxin.

In another embodiment of the present invention, the pharmaceuticalformulations are essentially free of salt. More preferably, theformulation contains less than about 0.9% salt.

In one embodiment of the present invention, the pharmaceuticalformulations have a pH of between about 5.6 to 6.0, about 6.0 to 6.4,about 6.4 to 6.8, about 6.8 to 7.2, about 5.8 to 7.4, about 6 to 7.4,about 6.2 to 7.4, about 6.5 to 7.4, about 6.7 to 7.4, about 7 to 7.4, orabout 7.2 to 7.4. Preferably, the pharmaceutical formulations have a pHof about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3,about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about7.0, about 7.1, about 7.2, about 7.3, or about 7.4.

The pharmaceutical formulations of the present invention may beadministered by any means known in the art sufficient to deliver thebotulinum toxin to the desired therapeutic target. Preferably, thepharmaceutical formulations are delivered by transmucosaladministration, transcutaneous administration, intramuscularadministration or topically. Preferably, the pharmaceutical formulationsof the present invention are administered by injection.

The pharmaceutical formulations of the present invention may be used inany of the methods of treatment disclosed herein. According to theinventive methods described herein, the pharmaceutical formulations ofthe present invention may be administered as a single treatment orrepeated periodically to provide multiple treatments.

The present invention also provides methods for muscle denervationcomprising the step of administering any of the pharmaceuticalformulations of the present invention to a subject in need thereof in anamount sufficient to produce local muscle denervation. In anotherembodiment, the pharmaceutical formulations are administered to themuscles of a head, face, eye, neck, back, or tissues overlying one ormore nasal sinuses.

In another embodiment, the present invention provides methods treatingneuromuscular diseases comprising the step of administering any of thepharmaceutical formulations of the present invention to a subject inneed thereof in an amount sufficient to produce muscle weakness. Inanother embodiment, the neuromuscular disease is cervical dystonia,hemifacial spasm, bruxism, blepharospasm, strabismus, or musclespasticity. In a preferred embodiment, the neuromuscular diseasehemifacial spasm, cervical dystonia, blepharospasm, strabismus, ormuscle spasticity.

In a preferred embodiment, the neuromuscular disease is hemifacialspasm. A subject suffering from hemifacial spasm preferably receivesbetween about 1.5 to 15 Units per treatment of any of the pharmaceuticalformulations of the present invention. More preferably, between about1.5 to 3 Units, 1.5 to 5 Units, 1.5 to 7 Units, 1.5 to 10 Units, 1.5 to12 Units, 1.5 to 15 Units, 5 to 10 Units, 5 to 15 Units, or 10 to 15Units per treatment are administered to a patient with hemifacial spasm.Most preferably, about 1.5, about 2, about 2.5, about 3, about 3.5,about 4, about 4.5 about 5, about 5.5, about 6, about 6.5, about 7,about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5,about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about14, about 14.5, or about 15 Units per treatment are administered to apatient with hemifacial spasm. Dosages greater than 15 Units pertreatment may also be administered to patients with hemifacial spasm toachieve a therapeutic response.

In a preferred embodiment, the neuromuscular disease is cervicaldystonia. A subject suffering from cervical dystonia preferably receivesbetween about 15 to 150 Units per treatment of any of the pharmaceuticalformulations of the present invention. More preferably, between about 15to 30 Units, 15 to 50 Units, 15 to 75 Units, 15 to 100 Units, 15 to 125Units, 15 to 150 Units, 20 to 100 Units, 20 to 150 Units, or 100 to 150Units per treatment are administered to a patient with cervicaldystonia. Most preferably, about 15, about 20, about 25, about 30, about35, about 40, about 45 about 50, about 55, about 60, about 65, about 70,about 75, about 80, about 85, about 90, about 95, about 100, about 105,about 110, about 115, about 120, about 125, about 130, about 135, about140, about 145, or about 150 Units per treatment are administered to apatient with cervical dystonia. Dosages greater than 150 Units pertreatment may also be administered to patients with cervical dystonia toachieve a therapeutic response.

In a preferred embodiment, the neuromuscular disease is blepharospasm. Asubject suffering from blepharospasm preferably receives between about1.5 to 20 Units per treatment of any of the pharmaceutical formulationsof the present invention. More preferably, between about 1.5 to 5 Units,1.5 to 7 Units, 1.5 to 10 Units, 1.5 to 12 Units, 1.5 to 15 Units, 1.5to 17 Units, 2.0 to 5 Units, 2 to 10 Units, 2 to 15, 2 to 20, 5 to 10, 5to 15, or 5 to 20 Units per treatment are administered to a patient withblepharospasm. Most preferably, about 1.5, about 2.0, about 2.5, about3.0, about 3.5, about 4.0, about 4.5 about 5.0, about 5.5, about 6.0,about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about9.5, about 10.0, about 10.5, about 11.0, about 11.5, about 12.0, about12.5, about 13.0, about 13.5, about 14.0, about 14.5, about 15.0, about15.5, about 16.0, about 16.5, about 17.0, about 17.5, about 18.0, about18.5, about 19.0, about 19.5, or about 20.0 Units per treatment areadministered to a patient with blepharospasm. Dosages greater than 20Units per treatment may also be administered to patients withblepharospasm to achieve a therapeutic response.

In a preferred embodiment, the neuromuscular disease is strabismus. Asubject suffering from strabismus preferably receives between about 4 to40 Units per treatment of any of the pharmaceutical formulations of thepresent invention. More preferably, between about 4 to 10 Units, 4 to 15Units, 4 to 20 Units, 4 to 25 Units, 4 to 30 Units, 4 to 35 Units, 7 to15 Units, 7 to 20 Units, 7 to 25, 7 to 30, 7 to 35, or 7 to 40 Units pertreatment are administered to a patient with strabismus. Mostpreferably, about 4, about 5, about 7.5, about 10, about 12.5, about 15,about 17.5 about 20.0, about 22.5, about 25.0, about 27.5, about 30.0,about 32.5, about 35, about 37.5, or about 40 Units per treatment areadministered to a patient with strabismus. Dosages greater than 40 Unitsper treatment may also be administered to patients with strabismus toachieve a therapeutic response.

In a preferred embodiment, the neuromuscular disease is musclespasticity. A subject suffering from muscle spasticity preferablyreceives between about 20 to 200 Units per treatment of any of thepharmaceutical formulations of the present invention. More preferably,between about 20 to 30 Units, 20 to 40 Units, 20 to 60 Units, 20 to 80Units, 20 to 100 Units, 20 to 125 Units, 20 to 150 Units, or 20 to 175Units per treatment are administered to a patient with musclespasticity. Most preferably, about 20, about 25, about 30, about 35,about 40, about 45 about 50, about 55, about 60, about 65, about 70,about 75, about 80, about 85, about 90, about 95, about 100, about 105,about 110, about 115, about 120, about 125, about 130, about 135, about140, about 145, about 150, about 155, about 160, about 165, about 170,about 175, about 180, about 185, about 190, about 195, or about 200Units per treatment are administered to a patient with musclespasticity. Dosages greater than 200 Units per treatment may also beadministered to patients with muscle spasticity to achieve a therapeuticresponse.

In another embodiment, the present invention provides methods fortreating pain comprising the step of administering any of thepharmaceutical formulations of the present invention to a subject inneed thereof in an amount sufficient to reduce pain. In anotherembodiment, the patient suffers from myofascial pain, migraine headachepain, tension headache pain, neuropathic pain, facial pain, lower-backpain, sinus-headache pain, pain associated with temporomandibular jointdisease, pain associated with spasticity or cervical dystonia,post-surgical wound pain, or neuralgia.

In a preferred embodiment, the patient suffers from sinus-headache pain.A subject suffering from sinus-headache pain preferably receives betweenabout 4 to 40 Units per treatment of any of the pharmaceuticalformulations of the present invention. More preferably, between about 4to 10 Units, 4 to 15 Units, 4 to 20 Units, 4 to 25 Units, 4 to 30 Units,4 to 35 Units, 7 to 15 Units, 7 to 20 Units, 7 to 25, 7 to 30, 7 to 35,or 7 to 40 Units per treatment are administered to a patient sufferingfrom sinus-headache pain. Most preferably, about 4, about 5, about 7.5,about 10, about 12.5, about 15, about 17.5 about 20.0, about 22.5, about25.0, about 27.5, about 30.0, about 32.5, about 35, about 37.5, or about40 Units per treatment are administered to a patient with sinus-headachepain. Dosages greater than 40 Units per treatment may also beadministered to patients with sinus headache-pain to achieve atherapeutic response.

In a preferred embodiment, the patient suffers from facial pain. Asubject suffering from facial pain preferably receives between about 4to 40 Units per treatment of any of the pharmaceutical formulations ofthe present invention. More preferably, between about 4 to 10 Units, 4to 15 Units, 4 to 20 Units, 4 to 25 Units, 4 to 30 Units, 4 to 35 Units,7 to 15 Units, 7 to 20 Units, 7 to 25, 7 to 30, 7 to 35, or 7 to 40Units per treatment are administered to a patient suffering from facialpain. Most preferably, about 4, about 5, about 7.5, about 10, about12.5, about 15, about 17.5 about 20.0, about 22.5, about 25.0, about27.5, about 30.0, about 32.5, about 35, about 37.5, or about 40 Unitsper treatment are administered to a patient with facial pain. Dosagesgreater than 40 Units per treatment may also be administered to patientswith facial pain to achieve a therapeutic response.

In a preferred embodiment, the patient suffers from myofascial pain. Asubject suffering from myofascial pain preferably receives between about5 to 100 Units per treatment of any of the pharmaceutical formulationsof the present invention. More preferably, between about 5 to 10 Units,5 to 20 Units, 5 to 30 Units, 5 to 40 Units, 5 to 50 Units, 5 to 60Units, 5 to 70 Units, 5 to 80 Units, 5 to 90, 10 to 20, 10 to 30, 10 to50 or 10 to 60, or 10 to 70, or 10 to 80, 10 to 90 or 10 to 100 Unitsper treatment are administered to a patient suffering from myofascialpain. Most preferably, about 5, about 10, about 15, about 20, about 25,about 30, about 35 about 40, about 45, about 50, about 55, about 60,about 65, about 70, about 75, about 80, about 85, about 90, about 95 orabout 100 Units per treatment are administered to a patient withmyofascial pain. Dosages greater than 100 Units per treatment may alsobe administered to patients with myofascial pain to achieve atherapeutic response.

In a preferred embodiment, the patient suffers from migraine-headachepain. A subject suffering from migraine-headache pain preferablyreceives between about 0.5 to 50 Units per treatment of any of thepharmaceutical formulations of the present invention. More preferably,between about 0.5 to 5 Units, 0.5 to 10 Units, 0.5 to 15 Units, 0.5 to20 Units, 0.5 to 25 Units, 0.5 to 30 Units, 0.5 to 35 Units, 0.5 to 40Units, 0.5 to 45, 0.5 to 50, 2 to 10, 2 to 20, 2 to 30, 2 to 40, or 2 to50 Units per treatment are administered to a patient suffering frommigraine-headache pain. Most preferably, about 0.5, about 1.0, about1.5, about 2.0, about 2.5, about 3.0, about 3.5 about 4.0, about 4.5,about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 12, about 15,about 17, about 20, about 22, about 25, about 27, about 30, about 32,about 35, about 37, about 40, about 42, about 45, about 47, or about 50Units per treatment are administered to a patient with migraine-headachepain. Dosages greater than 50 Units per treatment may also beadministered to patients with migraine-headache pain to achieve atherapeutic response.

In a preferred embodiment, the suffers from lower-back pain. A subjectsuffering from lower-back pain preferably receives between about 15 to150 Units per treatment of any of the pharmaceutical formulations of thepresent invention. More preferably, between about 15 to 30 Units, 15 to50 Units, 15 to 75 Units, 15 to 100 Units, 15 to 125 Units, 15 to 150Units, 20 to 100 Units, 20 to 150 Units, or 100 to 150 Units pertreatment are administered to a patient with lower-back pain. Mostpreferably, about 15, about 20, about 25, about 30, about 35, about 40,about 45 about 50, about 55, about 60, about 65, about 70, about 75,about 80, about 85, about 90, about 95, about 100, about 105, about 110,about 115, about 120, about 125, about 130, about 135, about 140, about145, or about 150 Units per treatment are administered to a patient withlower-back pain. Dosages greater than 150 Units per treatment may alsobe administered to patients with lower-back pain to achieve atherapeutic response.

In a preferred embodiment, the patient suffers from tension-headachepain. A subject suffering from tension-headache pain preferably receivesbetween about 5 to 50 Units per treatment of any of the pharmaceuticalformulations of the present invention. More preferably, between about 5to 10 Units, 5 to 15 Units, 5 to 20 Units, 5 to 25 Units, 5 to 30 Units,5 to 35 Units, 5 to 40 Units, 5 to 45 Units, 10 to 20, 10 to 25, 10 to30, 10 to 35, 10 to 40, 10 to 45, or 10 to 45 Units per treatment areadministered to a patient with tension-headache pain. Most preferably,about 5, about 10, about 20, about 25, about 30, about 35, about 40,about 45, or about 50 Units per treatment are administered to a patientwith tension-headache pain. Dosages greater than 50 Units per treatmentmay also be administered to patients with tension-headache pain toachieve a therapeutic response.

In a preferred embodiment, the patient suffers from sinus headache painor facial pain associated with acute or recurrent chronic sinusitis.Preferably, any of the pharmaceutical formulations of the presentinvention may be administered to the nasal mucosa or to the subcutaneousstructures overlying the sinuses, wherein the administration of theformulation reduces the headache and/or facial pain associated withacute recurrent or chronic sinusitis. More preferably, any of thepharmaceutical formulations of the present invention may be administeredto the nasal mucosa. The subcutaneous structures overlying the sinusespreferably overly one or more of the sinuses selected from the groupconsisting of: ethmoid; maxillary; mastoid; frontal; and sphenoid. Inanother embodiment, subcutaneous structures overlying the sinuses liewithin one or more of the areas selected from the group consisting of:forehead; malar; temporal; post auricular; and lip.

In another embodiment, a patient suffering from sinus headache pain orfacial pain associated with acute or recurrent chronic sinusitis istreated by administering any of the pharmaceutical formulations of thepresent invention to an afflicted area of the patient. In a preferredembodiment, the pharmaceutical formulations disclosed herein areadministered to the projections of a trigeminal nerve innervating asinus.

Patients suffering from sinus headache pain or facial pain associatedwith acute or recurrent chronic sinusitis often exhibit symptomsincluding rhinitis, sinus hypersecretion and/or purulent nasaldischarge. In one embodiment, the patients treated with thepharmaceutical formulations of the present invention exhibit symptoms ofsinus hypersecretion and purulent nasal discharge.

The present invention also provides methods for treating a patientsuffering from sinus headache pain or facial pain associated with acuteor recurrent chronic sinusitis, wherein the subject suffers fromneuralgia. Preferably, the neuralgia is trigeminal neuralgia. In anotherembodiment, the neuralgia is: associated with compressive forces on asensory nerve; associated with intrinsic nerve damage, demyelinatingdisease, or a genetic disorder; associated with a metabolic disorder;associated with central neurologic vascular disease; or associated withtrauma. In another embodiment of the present invention, the pain isassociated with dental extraction or reconstruction.

In another embodiment, the present invention provides methods forcosmetically modifying soft-tissue features comprising the step ofadministering any of the pharmaceutical formulations of the presentinvention to a subject in need thereof in an amount sufficient to modifysaid features. In a preferred embodiment, the pharmaceutical formulationis administered via transcutaneous or transmucosal injection either at asingle focus or multiple foci.

Preferably, the pharmaceutical formulations of the present invention areadministered to the face or neck of the subject. In a preferredembodiment, the pharmaceutical formulations of the present invention areadministered to the subject in an amount sufficient to reduce rhytides.Preferably, the formulation is administered between eyebrows of thesubject in an amount sufficient to reduce vertical lines between theeyebrows and on a bridge of a nose. The pharmaceutical formulations mayalso be administered near either one or both eyes of the subject in anamount sufficient to reduce lines at corners of the eyes. In anotherembodiment, the pharmaceutical formulations of the present invention mayalso be administered to a forehead of the subject in an amountsufficient to reduce horizontal lines on said forehead. In yet anotherembodiment of the present invention the pharmaceutical formulation isadministered to the neck of the subject in an amount sufficient toreduce muscle bands in the neck.

The present invention provides methods for reducing lip volume in one orboth of the upper and lower lips of a patient. In one embodiment, thepatient suffers from hypervolemic lip deformity. Preferably, thepharmaceutical formulations of the present invention are administered toa orbicularis oris muscle of the subject. The pharmaceutical botulinumtoxin formulations of the present invention may also be administered toone or more lip retractor muscle. A patient desiring the cosmeticreduction of lip volume or other soft-tissue structure, or the patientsuffering from hyper-volemic lip deformity preferably receives betweenabout 2 to 20 Units per treatment of any of the pharmaceuticalformulations of the present invention. More preferably, between about 2to 5 Units, 2 to 7 Units, 2 to 10 Units, 2 to 12 Units, 2 to 15 Units, 2to 20 Units, 5 to 10 Units, 5 to 15 Units, or 5 to 20 Units pertreatment are administered to a patient desiring the cosmetic reductionof lip volume or other soft-tissue structure, or the patient sufferingfrom hyper-volemic lip deformity. Most preferably, about 2, about 3,about 4, about 5, about 6, about 7, about 8 about 9, about 10, about 11,about 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19 or about 20 Units per treatment are administered to a patientdesiring the cosmetic reduction of lip volume or other soft-tissuestructure, or the patient suffering from hyper-volemic lip deformity.Dosages greater than 20 Units per treatment may also be administered topatient desiring the cosmetic reduction of lip volume or othersoft-tissue structure, or the patient suffering from hyper-volemic lipdeformity to achieve a therapeutic response.

In another embodiment, the present invention provides methods fortreating inflammation comprising the step of administering any of thepharmaceutical formulations of the present invention to a subject inneed thereof in an amount sufficient to reduce inflammation. Preferably,the pharmaceutical formulations of the present invention areadministered to a patient without producing muscle weakness. In oneembodiment, the pharmaceutical formulations of the present invention areadministered to patients with an inflammatory condition. Preferably, theinflammatory condition is neurogenic inflammation. In anotherembodiment, the subject suffers from rheumatoid arthritis or agastro-intestinal inflammatory disease.

In a preferred embodiment, the patient suffers from an inflammatorydisorder. A subject suffering from an inflammatory disorder preferablyreceives between about 1 to 100 Units per treatment of any of thepharmaceutical formulations of the present invention. More preferably,between about 1 to 10 Units, 1 to 20 Units, 1 to 30 Units, 1 to 40Units, 1 to 50 Units, 1 to 60 Units, 1 to 70 Units, 1 to 80 Units, 1 to90, 5 to 20, 5 to 30, 5 to 40, 5 to 50, 5 to 60, 5 to 70, 5 to 80, 5 to90, or 5 to 100 Units per treatment are administered to a patient withan inflammatory disorder. Most preferably, about 1, about 10, about 20,about 30, about 40, about 50, about 60, about 70, about 80, about 90, orabout 100 Units per treatment are administered to a patient withtension-headache pain. Dosages greater than 100 Units per treatment mayalso be administered to patients suffering from inflammation or aninflammatory disorder to achieve a therapeutic response.

In a preferred embodiment, the inflammatory disorder is blepharitis. Asubject suffering from blepharitis preferably receives between about 1to 10 Units per treatment of any of the pharmaceutical formulations ofthe present invention. More preferably, between about 1 to 3 Units,about 1 to 5 Units, about 1 to 7 Units, or 1 to 10 Units per treatmentare administered to a patient with an inflammatory disorder. Mostpreferably, about 1, about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, or about 10 Units per treatment are administered toa patient with tension-headache pain. Dosages greater than 10 Units pertreatment may also be administered to patients suffering frominflammation or an inflammatory disorder to achieve a therapeuticresponse.

In a preferred embodiment, the inflammatory disorder is prostatitis. Asubject suffering from prostatitis preferably receives between about 10to 100 Units per treatment of any of the pharmaceutical formulations ofthe present invention. More preferably, between about 10 to 20 Units,about 10 to 30 Units, about 10 to 40 Units, about 10 to 50 Units, about10 to 60 Units, about 10 to 70 Units, about 10 to 80 Units, or about 10to 90 Units per treatment are administered to a patient withprostatitis. Most preferably, about 10, about 20, about 30, about 40,about 50, about 60, about 70, about 80, about 90, or about 100 Units pertreatment are administered to a patient with prostatitis. Dosagesgreater than 100 Units per treatment may also be administered topatients with prostatitis to achieve a therapeutic response.

In another embodiment, the present invention provides methods fortreating cutaneous disorders comprising the step of administering any ofthe pharmaceutical formulations of the present invention to a subject inneed thereof in an amount sufficient to reduce a sebaceous or mucoussecretion. Preferably, the pharmaceutical formulations of the presentinvention are administered to a patient without producing muscleweakness. In one embodiment, the pharmaceutical formulations of thepresent invention are administered to patients with chalazion orhordeola. Preferably, the pharmaceutical formulations of the presentinvention are injected into one or more sites of an eyelid orconjunctiva. In another embodiment, the formulations of the presentinvention are administered to a body surface. In another embodiment, thepharmaceutical formulations are administered in an amount sufficient toreduce cutaneous bacterial or fungal growth, including but not limitedto Staphylococcus; Streptococcus and Moraxella. Preferably, thepharmaceutical formulations of the present invention are administered toan area selected from the group consisting of: eyelid; scalp; feet;groin; and armpit to reduce cutaneous infection.

In another embodiment, the cutaneous disorder is hyperhydrosis.

The present invention also provides methods for treating inflammationcomprising the step of administering any of the pharmaceuticalformulations of the present invention to a subject in need thereof in anamount sufficient to reduce inflammation. Preferably, the pharmaceuticalformulations of the present invention are administered to a patientwithout producing muscle weakness. In one embodiment, the pharmaceuticalformulations of the present invention are administered to patients withan inflammatory condition. Preferably, the inflammatory condition isneurogenic inflammation. In another embodiment, the subject suffers fromrheumatoid arthritis or a gastro-intestinal inflammatory disease.

In one embodiment, the present invention provides methods for treatingcervical dystonia comprising the step of administering between 150 to3500 picograms per treatment of a pharmaceutical formulation comprisinga botulinum toxin to a subject in need thereof in an amount sufficientto produces local muscle weakness in said subject. A subject in need ofmuscle denervation preferably receives between about 150 to 300picograms, about 150 to 400 picograms, about 150 to 500 picograms, about150 to 750 picograms, about 150 to 1000 picograms, about 150 to 1250picograms, about 150 to 1500 picograms, about 150 to 1750 picograms,about 150 to 2000 picograms, about 150 to 2250 picograms, about 150 to2500 picograms, about 150 to 2750 picograms, about 150 to 3000picograms, or about 150 to 3500 picograms of a pharmaceuticalformulation comprising a botulinum toxin per treatment. Most preferably,about 150, about 250, about 350, about 450, about 550, about 650, about750, about 850, about 950, about 1050, about 1150, about 1250, about1350, about 1450, about 1550, about 1650, about 1750, about 1850, about1950, about 2050, about 2150, about 2250, about 2350, about 2450, about2550, about 2650, about 2750, about 2850, about 2950, about 3000, about3100, about 3200, about 3300 about 3400 or about 3500 picograms pertreatment are administered to a subject to produce muscle denervation.Dosages greater than 3500 picograms per treatment may also beadministered to patients with cervical dystonia to achieve a therapeuticresponse.

The present invention also provides methods for treating blepharospasmcomprising the step of administering between about 2.5 to 45 picogramsper treatment of a pharmaceutical formulation comprising botulinum toxinto a subject in need thereof, wherein the administration of saidbotulinum toxin produces muscle weakness in said subject. A subject withblepharospasm preferably receives between about 2.5 to 5 picograms,about 2.5 to 7.5, about 2.5 to 10, about 2.5 to 12.5, about 2.5 to 15,about 2.5 to 17.5, about 2.5 to 20, about 2.5 to 25, about 2.5 to 30,about 2.5 to 35 about 2.5 to 40, about 2.5 to 45 picograms of apharmaceutical formulation comprising botulinum per treatment. Mostpreferably, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about5.0, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5,about 9.0 about 10, about 12, about 15, about 17, about 20, about 22,about 25, about 27, about 30, about 32, about 35, about 37, about 40,about 42, or about 45 picograms per treatment are administered to asubject to produce muscle weakness. Dosages greater than 45 picogramsper treatment may also be administered to patients with blepharospasm toachieve a therapeutic response.

The present invention also provides methods for treating strabismuscomprising the step of administering between about 2.5 to 45 picogramsper treatment of a pharmaceutical formulation comprising a botulinumtoxin to a subject in need thereof, wherein the administration of saidbotulinum toxin produces produce muscle weakness in said subject. Asubject with strabismus preferably receives between about 2.5 to 5picograms, about 2.5 to 7.5, about 2.5 to 10, about 2.5 to 12.5, about2.5 to 15, about 2.5 to 17.5, about 2.5 to 20, about 2.5 to 25, about2.5 to 30, about 2.5 to 35 about 2.5 to 40, about 2.5 to 45 picograms ofa pharmaceutical formulation comprising botulinum toxin per treatment.Most preferably, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5,about 5.0, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about8.5, about 9.0 about 10, about 12, about 15, about 17, about 20, about22, about, 25, about 27, about 30, about 32, about 35, about 37, about40, about 42, or about 45 picograms per treatment are administered to asubject to produce muscle weakness. Dosages greater than 45 picogramsper treatment may also be administered to patients with strabismus toachieve a therapeutic response.

The present invention also provides methods for treating musclespasticity comprising the step of administering between about 20 to 350picograms per treatment of a pharmaceutical formulation comprising abotulinum toxin to a subject in need thereof, wherein the administrationof said botulinum toxin produces produce muscle weakness in saidsubject. A subject with muscle spasticity preferably receives betweenabout 20 to 30 picograms, about 20 to 40 picograms, about 20 to 50picograms, about 20 to 60 picograms, about 20 to 70 picograms, about 20to 80 picograms, about 20 to 90 picograms, about 20 to 100 picograms,about 20 to 150 picograms, about 20 to 200 picograms, about 20 to 250picograms, about 20 to 300 or about 20 to 350 picograms of apharmaceutical formulation comprising a botulinum toxin per treatment.Most preferably, about 20, about 30, about 40, about 50, about 60, about70, about 80, about 90, about 100, about 110, about 120, about 130,about 140, about 150, about 160 about 170, about 180, about 190, about200, about 210, about, 220, about 230, about 240, about 250, about 260,about 270, about 280, about 290, about 300, about 310, about 320, about330, about 340, or about 350 picograms per treatment are administered toa subject to produce muscle weakness. Dosages greater than 350 picogramsper treatment may also be administered to patients with musclespasticity to achieve a therapeutic response.

In a preferred embodiment, the pharmaceutical formulation comprising abotulinum toxin is administered to a subject suffering muscle spasticityin the flexor digitorum profundus muscle or the flexor digitorumsublimus muscle.

In another embodiment between about 20 to 450 picograms per treatment ofa pharmaceutical formulation comprising a botulinum toxin isadministered to a subject suffering muscle spasticity to produce muscleweakness in the flexor carpii ulnaris muscle. Preferably between about20 to 50 picograms, about 20 to 75 picograms, about 20 to 100 picograms,about 20 to 125 picograms, about 20 to 150 picograms, about 20 to 175picograms, about 20 to 200 picograms, about 20 to 225 picograms, about20 to 250 picograms, about 20 to 275 picograms, about 20 to 300picograms, about 20 to 325 picograms, about 20 to 350 picograms, about20 to 375 picograms, about 20 to 400 picograms, about 20 to 425picograms, or about 20 to 450 picograms of a pharmaceutical formulationcomprising a botulinum toxin are administered per treatment to a subjectin need thereof. Most preferably, about 20, about 30, about 40, about50, about 60, about 70, about 80, about 90, about 100, about 110, about120, about 130, about 140, about 150, about 160 about 170, about 180,about 190, about 200, about 210, about, 220, about 230, about 240, about250, about 260, about 270, about 280, about 290, about 300, about 310,about 320, about 330, about 340, about 350, about 360, about 370, about380, about 390, about 400, about 410, about 420, about 430 about 440, orabout 450 picograms per treatment are administered to a subject toproduce muscle weakness in the flexor carpii ulnaris muscle. Dosagesgreater than 450 picograms per treatment may also be administered topatients with muscle spasticity to achieve a therapeutic response.

In another embodiment between about 35 to 725 picograms per treatment ofthe pharmaceutical formulation comprising a botulinum toxin isadministered to a subject suffering muscle spasticity to produce muscleweakness in the flexor carpii radialis muscle. Preferably between about35 to 50 picograms, about 35 to 75 picograms, about 35 to 100 picograms,about 35 to 125 picograms, about 35 to 150 picograms, about 35 to 175picograms, about 35 to 200 picograms, about 35 to 225 picograms, about35 to 250 picograms, about 35 to 275 picograms, about 35 to 300picograms, about 35 to 325 picograms, about 35 to 350 picograms, about35 to 375 picograms, about 35 to 400 picograms, about 35 to 425picograms, about 35 to 450, about 35 to 475, about 35 to 500, about 35to 525, about 35 to 550, about 35 to 575, about 35 to 600, about 35 to625, about 35 to 650, about 35 to 675, about 35 to 700, about 35 to 725,or about 35 to 750, picograms of a pharmaceutical formulation comprisinga botulinum toxin are administered per treatment to a subject in needthereof. Most preferably, about 35, about 45, about 55, about 65, about75, about 85, about 95, about 100, about 110, about 120, about 130,about 140, about 150, about 160 about 170, about 180, about 190, about200, about 210, about, 220, about 230, about 240, about 250, about 260,about 270, about 280, about 290, about 300, about 310, about 320, about330, about 340, about 350, about 360, about 370, about 380, about 390,about 400, about 410, about 420, about 430, about 440, about 450, about460, about 470, about 480, about 490, about 500, about 510, about 520,about 530, about 540, about 550, about 560, about 570, about 580, about590, about 600, about 610, about 620, about 630, about 640, about 650,about 660, about 670, about 680, about 690, about 700, about 710, about720, or about 725 picograms per treatment are administered to a subjectto produce muscle weakness in the flexor carpii radialis muscle. Dosagesgreater than 725 picograms per treatment may also be administered topatients with muscle spasticity to achieve a therapeutic response.

In another embodiment between about 100 to 2250 picograms per treatmentof the pharmaceutical formulation comprising a botulinum toxin isadministered to a subject suffering muscle spasticity to produce muscleweakness in the biceps brachii muscle. Preferably between about 100 to125 picograms, about 100 to 150 picograms, about 100 to 175 picograms,about 100 to 200 picograms, about 100 to 250 picograms, about 100 to 300picograms, about 100 to 350 picograms, about 100 to 400 picograms, about100 to 450 picograms, about 100 to 500 picograms, about 100 to 600picograms, about 100 to 700 picograms, about 100 to 800 picograms, about100 to 900 picograms, about 100 to 1000 picograms, about 100 to 1100picograms, about 100 to 1200 picograms, about 100 to 1300 picograms,about 100 to 1400 picograms, about 100 to 1500 picograms, about 100 to1600 picograms, about 100 to 1700 picograms, about 100 to 1800picograms, about 100 to 1900 picograms, about 100 to 2000 picograms,about 100 to 2100 picograms, or about 100 to 2250 picograms of apharmaceutical formulation comprising a botulinum toxin is administeredper treatment to a subject in need thereof. Most preferably, about 100,about 150, about 200, about 250, about 300, about 350, about 400, about450, about 500, about 550, about 600, about 650, about 700, about 750about 800, about 850, about 900, about 950, about 1000, about, 1050,about 1100, about 1150, about 1200, about 1250, about 1300, about 1350,about 1400, about 1450, about 1500, about 1550, about 1600, about 1650,about 1700, about 1750, about 1800, about 1850, about 1900, about 1950,about 2000, about 2050, about 2100, about 2150, about 2200, or about2250 picograms per treatment are administered to a subject to producemuscle weakness in the biceps brachii muscle. Dosages greater than 2250picograms per treatment may also be administered to patients with musclespasticity to achieve a therapeutic response.

The present invention also provides methods for treating pain and painsyndromes comprising the step of administering between about 1.0 to 20picograms per treatment of a pharmaceutical composition comprising abotulinum toxin to a subject in need thereof, wherein the administrationof said botulinum toxin formulation reduces pain in said subject.Preferably, a patient suffering from pain or a pain syndrome receivesbetween about 1 to 3, about 1 to 5, about 1 to 7, about 1 to 10, about 1to 12, about 1 to 15, about 1 to 17, about 5 to 10, about 5 to 15, about5 to 20, or about 1 to 20 picograms per treatment of a pharmaceuticalcomposition comprising botulinum toxin. Most preferably, about 1, about2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about10, about 11, about 12, about 13, about 14, about 15, about 16, about17, about 18, about 19, or about 20 picograms per treatment areadministered to a subject to reduce pain. Dosages greater than 20picograms per treatment may also be administered to patients with musclespasticity to achieve a therapeutic response.

The present invention also provides methods for cosmetically modifyingsoft-tissue features comprising the step of administering between about5 to 70 picograms per treatment of a pharmaceutical formulationcomprising a botulinum toxin to a subject in need thereof, wherein theadministration of said botulinum toxin is sufficient to modify saidfeatures in said subject. Preferably, a patient desiring to modify softtissue features receives between about 5 to 10, about 5 to 15, about 5to 20, about 5 to 25, about 5 to 30, about 5 to 35, about 5 to 40, about5 to 45, about 5 to 55, about 5 to 60, about 5 to 65 or about 5 to 75picograms per treatment of a pharmaceutical composition comprisingbotulinum toxin. Most preferably, about 5, about 7, about 10, about 12,about 15, about 17, about 20, about 22, about 25, about 27, about 30,about 32, about 35, about 37, about 40, about 42, about 45, about 47,about 50, about 52, about 55, about 57, about 60, about 62, about 65,about 67, or about 70 picograms per treatment are administered to asubject to modify soft tissue features. Dosages greater than 70picograms per treatment may also be administered to achieve atherapeutic response.

The present invention also provides methods for treating inflammationcomprising the step of administering between about 1.0 to 20 picogramsper treatment of a botulinum toxin to a subject in need thereof, whereinthe administration of said botulinum toxin reduces inflammation in saidsubject. Preferably, a patient suffering from inflammation receivesbetween about 1 to 2, about 1 to 5, about 1 to 7, about 1 to 10, about 1to 12, about 1 to 15, about 1 to 17, or about 1 to 20 picograms pertreatment of a pharmaceutical composition comprising a botulinum toxin.Most preferably, about 1, about 2, about 3, about 4, about 5, about 6,about 7, about 8, about 9, about 10, about 11, about 12, about 13, about14, about 15, about 16, about 17, about 18, about 19, or about 20picograms per treatment are administered to a subject to reduceinflammation. Dosages greater than 20 picograms per treatment may alsobe administered to reduce inflammation.

The present invention also provides methods of treating cutaneousdisorders comprising the step of administering between about 1.0 to 10picograms per treatment of a botulinum toxin to a subject in needthereof, wherein the administration of said botulinum toxin reduces asebaceous, meibomian or mucous secretion in said subject. Preferably, apatient suffering from cutaneous disorders receives between about 1 to2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7,about 1 to 8, about 1 to 9, or about 1 to 10 picograms per treatment ofa pharmaceutical composition comprising a botulinum toxin. Mostpreferably, about 1, about 2, about 3, about 4, about 5, about 6, about7, about 8, about 9, or about 10 picograms per treatment areadministered to a subject to reduce a sebaceous, meibomian or mucoussecretion. Dosages greater than 10 picograms per treatment may also beadministered to reduce sebaceous, meibomian or mucous secretions.

The present invention also provides methods for producing a high-potencybotulinum toxin formulation comprising the step of adding greater thanabout 500 micrograms of a sequestration agent per 100 LD₅₀ Units of abotulinum neurotoxin, wherein said formulation has an increased clinicalpotency. Preferably, greater than about 550, greater than about 600,greater than about 650, greater than about 700, greater than about 750,greater than about 800, greater than about 850, greater than about 900,greater than about 950, greater than about 1000, greater than about1500, greater than about 2000, greater than about 2500, greater thanabout 3000, greater than about 3500, greater than about 4000, greaterthan about 4500, greater than about 5000, greater than about 5500,greater than about 6000, greater than about 6500, greater than about7000, greater than about 7500, greater than about 8000, greater thanabout 8500, greater than about 9000, greater than about 9500, greaterthan about 10,000, greater than about 15,000, greater than about 20,000,greater than about 25,000, greater than about 50,000, greater than about100,000, greater than about 150,000, greater than about 200,000 orgreater than about 250,000 micrograms of a sequestration agent is addedper 100 LD₅₀ Units of a botulinum neurotoxin. More preferably, betweenabout 500 to 1000, about 500 to 1500, about 500 to 2000, about 500 to2500, about 500 to 3000, about 500 to 3500, about 500 to 4000, about 500to 4500, about 500 to 5000, about 500 to 10,000, about 500 to 20,000,about 1000 to 1500, about 1000 to 2000, about 1000 to 2500, about 1000to 3000, about 1000 to 3500, about 1000 to 4000, about 1000 to 4500,about 1000 to 5000, about 1000 to 5500, about 1000 to 6000, about 1000to 6500, about 1000 to 7000, about 1000 to 7500, about 1000 to 8000,about 1000 to 8500, about 1000 to 9000, about 1000 to 9500, about 1000to 10,000, about 1000 to 15,000, about 1000 to 20,000, about 1000 to25,000, or about 1000 to 50,000 micrograms of a sequestration agent isadded to 100 LD₅₀ Units o a botulinum neurotoxin, and wherein theresultant formulation has an increased clinical potency.

The present invention also provides methods of treating cervicaldystonia comprising the step of administering between about 15 to 150Units per treatment of a pharmaceutical formulation comprising abotulinum toxin to a subject in need thereof, wherein administration ofsaid formulation produces muscle weakness. In a preferred embodiment,the pharmaceutical formulation is administered to one or more of themuscles selected from the group consisting of: sternomastoid, levatorscapulae, splenius cervis, capitus, scalene, and trapezius. Preferably,between about 15 to 30 Units, 15 to 50 Units, 15 to 75 Units, 15 to 100Units, 15 to 125 Units, 15 to 150 Units, 20 to 100 Units, 20 to 150Units, or 100 to 150 Units per treatment are administered to a patientwith cervical dystonia. Most preferably, about 15, about 20, about 25,about 30, about 35, about 40, about 45 about 50, about 55, about 60,about 65, about 70, about 75, about 80, about 85, about 90, about 95,about 100, about 105, about 110, about 115, about 120, about 125, about130, about 135, about 140, about 145, or about 150 Units per treatmentare administered to a patient with cervical dystonia. Dosages greaterthan 150 Units per treatment may also be administered to patients withcervical dystonia to achieve a therapeutic response.

In another embodiment, the present invention provides methods oftreating blepharospasm comprising the step of administering betweenabout 1.5 to 20 Units per treatment of a pharmaceutical formulationcomprising a botulinum toxin to a subject in need thereof, whereinadministration of said formulation produces muscle weakness. Preferably,the pharmaceutical formulation is administered to an orbicular muscle ofsaid subject. In another embodiment, between about 1.5 to 5 Units, 1.5to 7 Units, 1.5 to 10 Units, 1.5 to 12 Units, 1.5 to 15 Units, 1.5 to 17Units, 2.0 to 5 Units, 2 to 10 Units, 2 to 15, 2 to 20, 5 to 10, 5 to15, or 5 to 20 Units per treatment are administered to a patient withblepharospasm. Most preferably, about 1.5, about 2.0, about 2.5, about3.0, about 3.5, about 4.0, about 4.5 about 5.0, about 5.5, about 6.0,about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about9.5, about 10.0, about 10.5, about 11.0, about 11.5, about 12.0, about12.5, about 13.0, about 13.5, about 14.0, about 14.5, about 15.0, about15.5, about 16.0, about 16.5, about 17.0, about 17.5, about 18.0, about18.5, about 19.0, about 19.5, or about 20.0 Units per treatment areadministered to a patient with blepharospasm. Dosages greater than 20Units per treatment may also be administered to patients withblepharospasm to achieve a therapeutic response.

In yet another embodiment, the present invention provides methods oftreating hyperhydrosis comprising the step of administering betweenabout 0.5 to 50 Units per treatment of a pharmaceutical formulationcomprising a botulinum toxin to a subject in need thereof, whereinadministration of said formulation reduces sweating. Preferably, thepharmaceutical formulation is administered to an axillae. In anotherembodiment, between about 0.5 to 5 Units, 0.5 to 10 Units, 0.5 to 15Units, 0.5 to 20 Units, 0.5 to 25 Units, 0.5 to 30 Units, 0.5 to 35Units, 0.5 to 40 Units, 0.5 to 45, 0.5 to 50, 2 to 10, 2 to 20, 2 to 30,2 to 40, or 2 to 50 Units per treatment are preferably administered to apatient suffering from hyperhydrosis. Most preferably, about 0.5, about1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5 about 4.0,about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 12,about 15, about 17, about 20, about 22, about 25, about 27, about 30,about 32, about 35, about 37, about 40, about 42, about 45, about 47, orabout 50 Units per treatment are administered to a patient withhyperhydrosis. Dosages greater than 50 Units per treatment may also beadministered to patients with hyperhydrosis to achieve a therapeuticresponse.

The present invention also provides methods of treating migraineheadache pain comprising the step of administering between about 0.5 to50 Units per treatment of a pharmaceutical formulation comprising abotulinum toxin to a subject in need thereof, wherein administration ofsaid formulation reduces migraine headache pain. In another embodiment,between about 0.5 to 5 Units, 0.5 to 10 Units, 0.5 to 15 Units, 0.5 to20 Units, 0.5 to 25 Units, 0.5 to 30 Units, 0.5 to 35 Units, 0.5 to 40Units, 0.5 to 45, 0.5 to 50, 2 to 10, 2 to 20, 2 to 30, 2 to 40, or 2 to50 Units per treatment are preferably administered to a patientsuffering from migraine headache pain. Most preferably, about 0.5, about1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5 about 4.0,about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 12,about 15, about 17, about 20, about 22, about 25, about 27, about 30,about 32, about 35, about 37, about 40, about 42, about 45, about 47, orabout 50 Units per treatment are administered to a patient with migraineheadache pain. Dosages greater than 50 Units per treatment may also beadministered to patients with migraine headache pain to achieve atherapeutic response.

The present invention also provides methods of treating facial paincomprising the step of administering between about 4 to 40 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationreduces facial pain. In one embodiment, the facial pain is associatedwith sinusitis. In another embodiment, the facial pain is associatedwith trigeminal neuralgia. In yet another embodiment, the facial pain ispost-surgical wound pain. In another embodiment, between about 4 to 7Units, about 4 to 10 Units, about 4 to 15 Units, about 4 to 20 Units,about 4 to 25 Units, about 4 to 30 Units, about 4 to 35 Units, or about4 to 40 Units per treatment are preferably administered to a patientsuffering from facial pain. Most preferably, about 4.0, about 4.5, about5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0,about 8.5, about 9.0, about 9.5, about 10.0, about 12, about 15, about17, about 20, about 22, about 25, about 27, about 30, about 32, about35, about 37, about 40, about 42, about 45, about 47, or about 50 Unitsper treatment are administered to a patient with facial pain. Dosagesgreater than 40 Units per treatment may also be administered to patientswith facial pain to achieve a therapeutic response.

The present invention also provides methods of treating strabismuscomprising the step of administering between about 4 to 40 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationreduces symptoms of strabismus. Preferably, between about 4 to 7 Units,about 4 to 10 Units, about 4 to 15 Units, about 4 to 20 Units, about 4to 25 Units, about 4 to 30 Units, about 4 to 35 Units, or about 4 to 40Units per treatment are preferably administered to a patient sufferingfrom strabismus. Most preferably, about 4.0, about 4.5, about 5.0, about5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5,about 9.0, about 9.5, about 10.0, about 12, about 15, about 17, about20, about 22, about 25, about 27, about 30, about 32, about 35, about37, about 40, about 42, about 45, about 47, or about 50 Units pertreatment are administered to a patient with strabismus. Dosages greaterthan 40 Units per treatment may also be administered to patients withstrabismus to achieve a therapeutic response.

The present invention also provides methods of treating hyperactivebladder comprising the step of administering between about 4 to 40 Unitsper treatment of a pharmaceutical formulation comprising a botulinumtoxin to a subject in need thereof, wherein administration of saidformulation reduces urination frequency. Preferably, between about 4 to7 Units, about 4 to 10 Units, about 4 to 15 Units, about 4 to 20 Units,about 4 to 25 Units, about 4 to 30 Units, about 4 to 35 Units, or about4 to 40 Units per treatment are preferably administered to a patientsuffering from hyperactive bladder. Most preferably, about 4.0, about4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5,about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 12, about15, about 17, about 20, about 22, about 25, about 27, about 30, about32, about 35, about 37, about 40, about 42, about 45, about 47, or about50 Units per treatment are administered to a patient with hyperactivebladder. Dosages greater than 40 Units per treatment may also beadministered to patients with facial pain to achieve a therapeuticresponse.

The present invention also provides methods of treating musclespasticity comprising the step of administering between about 20 to 200Units per treatment of a pharmaceutical formulation comprising abotulinum toxin to a subject in need thereof, wherein administration ofsaid formulation produces muscle weakness. Preferably, between about 20to 30 Units, 20 to 40 Units, 20 to 60 Units, 20 to 80 Units, 20 to 100Units, 20 to 125 Units, 20 to 150 Units, or 20 to 175 Units pertreatment are administered to a patient with muscle spasticity. Mostpreferably, about 20, about 25, about 30, about 35, about 40, about 45about 50, about 55, about 60, about 65, about 70, about 75, about 80,about 85, about 90, about 95, about 100, about 105, about 110, about115, about 120, about 125, about 130, about 135, about 140, about 145,about 150, about 155, about 160, about 165, about 170, about 175, about180, about 185, about 190, about 195, or about 200 Units per treatmentare administered to a patient with muscle spasticity. Dosages greaterthan 200 Units per treatment may also be administered to patients withmuscle spasticity to achieve a therapeutic response.

The present invention also provides methods of treating hemifacial spasmcomprising the step of administering between about 1.5 to 15 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationproduces muscle weakness. Preferably, between about 1.5 to 5 Units,about 1.5 to 7 Units, about 1.5 to 10 Units, about 1.5 to 12 Units,about 1.5 to 15 Units, about 2.0 to 5 Units, about 2 to 10 Units, about2 to 15, about 5 to 10, about 5 to 15, or about 5 to 20 Units pertreatment are administered to a patient with hemifacial spasm. Mostpreferably, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about4.0, about 4.5 about 5.0, about 5.5, about 6.0, about 6.5, about 7.0,about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about10.5, about 11.0, about 11.5, about 12.0, about 12.5, about 13.0, about13.5, about 14.0, about 14.5, or about 15.0 Units per treatment areadministered to a patient with hemifacial spasm. Dosages greater than 15Units per treatment may also be administered to patients with hemifacialspasm to achieve a therapeutic response.

The present invention also provides methods of myofascial paincomprising the step of administering between about 5 to 100 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationreduces myofascial pain. In a preferred embodiment, the myofascial painis pain associated with cervical dystonia or temporal mandibular jointsyndrome. More preferably, the myofascial pain is pain associated withcervical dystonia. In another embodiment, between about 5 to 10 Units, 5to 20 Units, 5 to 30 Units, 5 to 40 Units, 5 to 50 Units, 5 to 60 Units,5 to 70 Units, 5 to 80 Units, 5 to 90, 10 to 20, 10 to 30, 10 to 50 or10 to 60, or 10 to 70, or 10 to 80, 10 to 90 or 10 to 100 Units pertreatment are administered to a patient suffering from myofascial pain.Most preferably, about 5, about 10, about 15, about 20, about 25, about30, about 35 about 40, about 45, about 50, about 55, about 60, about 65,about 70, about 75, about 80, about 85, about 90, about 95 or about 100Units per treatment are administered to a patient with myofascial pain.Dosages greater than 100 Units per treatment may also be administered topatients with myofascial pain to achieve a therapeutic response.

The present invention also provides methods of treating facial paincomprising the step of administering between about 4 to 40 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationreduces facial pain. Preferably, between about 4 to 10 Units, 4 to 15Units, 4 to 20 Units, 4 to 25 Units, 4 to 30 Units, 4 to 35 Units, 7 to15 Units, 7 to 20 Units, 7 to 25, 7 to 30, 7 to 35, or 7 to 40 Units pertreatment are administered to a patient suffering from facial pain. Mostpreferably, about 4, about 5, about 7.5, about 10, about 12.5, about 15,about 17.5 about 20.0, about 22.5, about 25.0, about 27.5, about 30.0,about 32.5, about 35, about 37.5, or about 40 Units per treatment areadministered to a patient with facial pain. Dosages greater than 40Units per treatment may also be administered to patients with facialpain to achieve a therapeutic response.

The present invention also provides methods of treating inflammationcomprising the step of administering between about 5 to 100 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationreduces inflammation. In a preferred embodiment, the inflammation isassociated with arthritis or the inflammation is intestinal. In anotherembodiment, between about 1 to 10 Units, 1 to 20 Units, 1 to 30 Units, 1to 40 Units, 1 to 50 Units, 1 to 60 Units, 1 to 70 Units, 1 to 80 Units,1 to 90, 5 to 20, 5 to 30, 5 to 40, 5 to 50, 5 to 60, 5 to 70, 5 to 80,5 to 90, or 5 to 100 Units per treatment are administered to a patientwith an inflammatory disorder. Most preferably, about 1, about 10, about20, about 30, about 40, about 50, about 60, about 70, about 80, about90, or about 100 Units per treatment are administered to a patient withtension-headache pain. Dosages greater than 100 Units per treatment mayalso be administered to patients suffering from inflammation or aninflammatory disorder to achieve a therapeutic response.

The present invention also provides methods of method of treatingblepharitis comprising the step of administering between about 1 to 10Units per treatment of a pharmaceutical formulation comprising abotulinum toxin to a subject in need thereof, wherein administration ofsaid formulation reduces inflammation. In a preferred embodiment,between about 1 to 3 Units, about 1 to 5 Units, about 1 to 7 Units, or 1to 10 Units per treatment are administered to a patient with aninflammatory disorder. Most preferably, about 1, about 2, about 3, about4, about 5, about 6, about 7, about 8, about 9, or about 10 Units pertreatment are administered to a patient with tension-headache pain.Dosages greater than 10 Units per treatment may also be administered topatients suffering from inflammation or an inflammatory disorder toachieve a therapeutic response.

The present invention also provides methods of treating scoliosiscomprising the step of administering between about 30 to 300 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationimproves posture. In a preferred embodiment, between about 30 to 50Units, about 30 to 75 units, about 30 to 100 Units, about 30 to 125Units, about 30 to 150 Units, about 30 to 175 Units, about 30 to 200Units, about 30 to 225 Units, about 30 to 250 Units, about 30 to 275Units, or about 30 to 300 Units per treatment are administered to apatient with scoliosis. More preferably, about 30, about 35, about 40,about 45, about 50, about 55, about 60, about 65, about 70, about 75,about 80, about 85, about 90, about 100, about 110, about 120, about130, about 140, about 145, about 150, about 155, about 160, about 165,about 170, about 175, about 180, about 185, about 190, about 200, about210, about 220, about 230, about 240, about 245, about 250, about 255,about 260, about 265, about 270, about 275, about 280, about 285, about290, or about 300 Units per treatment are administered to a patient withscoliosis. Dosages greater than 300 Units per treatment may also beadministered to patients suffering from scoliosis to achieve atherapeutic response.

The present invention also provides methods of treating tension headachecomprising the step of administering between about 5 to 50 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationreduces pain. Preferably, between about 5 to 10 Units, 5 to 15 Units, 5to 20 Units, 5 to 25 Units, 5 to 30 Units, 5 to 35 Units, 5 to 40 Units,5 to 45 Units, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to45, or 10 to 45 Units per treatment are administered to a patient withtension-headache pain. Most preferably, about 5, about 10, about 20,about 25, about 30, about 35, about 40, about 45, or about 50 Units pertreatment are administered to a patient with tension-headache pain.Dosages greater than 50 Units per treatment may also be administered topatients with tension-headache pain to achieve a therapeutic response.

The present invention also provides methods of treating lower back paincomprising the step of administering between about 15 to 150 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationreduces pain. Preferably, between about 15 to 30 Units, 15 to 50 Units,15 to 75 Units, 15 to 100 Units, 15 to 125 Units, 15 to 150 Units, 20 to100 Units, 20 to 150 Units, or 100 to 150 Units per treatment areadministered to a patient with lower-back pain. Most preferably, about15, about 20, about 25, about 30, about 35, about 40, about 45 about 50,about 55, about 60, about 65, about 70, about 75, about 80, about 85,about 90, about 95, about 100, about 105, about 110, about 115, about120, about 125, about 130, about 135, about 140, about 145, or about 150Units per treatment are administered to a patient with lower-back pain.Dosages greater than 150 Units per treatment may also be administered topatients with lower-back pain to achieve a therapeutic response.

The present invention also provides methods of treating sclerodermacomprising the step of administering between about 30 to 300 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationreduces a symptom of scleroderma. In a preferred embodiment, betweenabout 30 to 50 Units, about 30 to 75 units, about 30 to 100 Units, about30 to 125 Units, about 30 to 150 Units, about 30 to 175 Units, about 30to 200 Units, about 30 to 225 Units, about 30 to 250 Units, about 30 to275 Units, or about 30 to 300 Units per treatment are administered to apatient with scleroderma. More preferably, about 30, about 35, about 40,about 45, about 50, about 55, about 60, about 65, about 70, about 75,about 80, about 85, about 90, about 100, about 110, about 120, about130, about 140, about 145, about 150, about 155, about 160, about 165,about 170, about 175, about 180, about 185, about 190, about 200, about210, about 220, about 230, about 240, about 245, about 250, about 255,about 260, about 265, about 270, about 275, about 280, about 285, about290, or about 300 Units per treatment are administered to a patient withscleroderma. Dosages greater than 300 Units per treatment may also beadministered to patients suffering from scleroderma to achieve atherapeutic response.

The present invention also provides methods of treating asthma and/orhayfever comprising the step of administering between about 5 to 50Units per treatment of a pharmaceutical formulation comprising abotulinum toxin to a subject in need thereof, wherein administration ofsaid formulation reduces inflammation. Preferably, between about 5 to 10Units, 5 to 15 Units, 5 to 20 Units, 5 to 25 Units, 5 to 30 Units, 5 to35 Units, 5 to 40 Units, 5 to 45 Units, 10 to 20, 10 to 25, 10 to 30, 10to 35, 10 to 40, 10 to 45, or 10 to 45 Units per treatment areadministered to a patient with asthma and/or hayfever. Most preferably,about 5, about 10, about 20, about 25, about 30, about 35, about 40,about 45, or about 50 Units per treatment are administered to a patientwith asthma and/or hayfever. Dosages greater than 50 Units per treatmentmay also be administered to patients with asthma and/or hayfever toachieve a therapeutic response.

The present invention also provides methods of treating prostatitiscomprising the step of administering between about 10 to 100 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxinto a subject in need thereof, wherein administration of said formulationreduces inflammation. In a preferred embodiment, the administration ofsaid formulation reduces prostate size. Preferably, between about 10 to30 Units, about 10 to 50 Units, about 10 to 75 Units, about 10 to 100Units, about 20 to 50 Units, about 20 to 75 Units, or about 20 to 100Units per treatment are administered to a patient with prostatitis. Mostpreferably, about 10, about 15, about 20, about 25, about 30, about 35,about 40, about 45 about 50, about 55, about 60, about 65, about 70,about 75, about 80, about 85, about 90, about 95, or about 100 Units pertreatment are administered to a patient with prostatitis. Dosagesgreater than 100 Units per treatment may also be administered topatients with prostatitis to achieve a therapeutic response.

The present invention also provides methods of treating facial rhytidescomprising the step of administering between about 2 to 20 Units pertreatment of a pharmaceutical formulation comprising a botulinum toxin asubject in need thereof, wherein administration of said formulationreduces facial lines. In a preferred embodiment, the pharmaceuticalformulation is administered to reduce vertical lines between theeyebrows and/or on a bridge of a nose. In another embodiment, thepharmaceutical formulation is administered to reduce lines at corners ofthe eyes. In yet another embodiment, the pharmaceutical formulation isadministered to reduce horizontal lines on said forehead. In a preferredembodiment, between about 2 to 5 Units, about 2 to 6 Units, about 2 to 7Units, about 2 to 8 Units, about 2 to 9 Units, about 2 to 10 Units,about 2 to 11 Units, about 2 to 12 Units, about 2 to 13 Units, about 2to 14 Units, about 2 to 15 Units, about 2 to 16 Units, about 2 to 17Units, about 1 to 18 Units, 1 to 19 Units, or about 2 to 20 Units pertreatment are administered to a patient with facial rhytides. Mostpreferably, about 2, about 3, about 4, about 5, about 6, about 7, about8, about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, or about 20 Units per treatmentare administered to a patient with facial rhytides. Dosages greater than20 Units per treatment may also be administered to a patient with facialrhytides to achieve a therapeutic response.

The present invention also provides methods for reducing scarring and/orcosmetic deformity associated with burns or skin disorders such asblistering dermatosis comprising the step of administering apharmaceutical formulation comprising a botulinum toxin to a subject inneed thereof, wherein administration of said formulation reducesscarring and/or cosmetic deformity. In another embodiment, thepharmaceutical formulation comprises a botulinum toxin and asequestration agent, as disclosed herein. In yet another embodiment, thepharmaceutical formulation comprises a botulinum toxin, a sequestrationagent, as disclosed herein, and a stabilizing agent, such as trehalose.Further, the pharmaceutical formulation comprises an agent that promotescutaneous absorption and penetration. In a preferred embodiment, thepharmaceutical formulation is administered to a body surface of saidsubject. In a preferred embodiment, the pharmaceutical formulation isadministered as a liquid formulation. More preferably, thepharmaceutical formulation is applied as an aerosol. In anotherembodiment, between about 1 and 10, about 1 and 50, about 1 and 100,about 1 and 200, about 1 and 500, about 1 and 1000, about 1 and 1250,about 1 and 1500, about 1 and 2000, or about 1 and 2500 Units pertreatment are administered to a subject suffering from a burn. As usedherein, “burn” includes but is not limited to thermal, electrical, orchemical burns and also includes blistering caused by dermatitis andother blistering disorders. In an alternative embodiment, mechanicalabrasion, chemical, thermal, laser-induced disruption of skin barriers,and the like, may be used to improve the delivery of topicaladministration of pharmaceutical formulations of botulinum toxin.

The present invention provides a composition comprising botulinum toxinand a sequestration agent for use in treating various neuromusculardiseases and localized denervation. In one embodiment, the sequestrationagent is present in an amount between 550 and 550,000 μg sequestrationagent per 100 LD₅₀ units botulinum toxin. In another embodiment, thesequestration agent is present in an amount between 550 and 5,500 μgsequestration agent per 100 LD₅₀ units botulinum toxin. In a furtherembodiment, the sequestration agent is present in an amount between5,500 and 13,000 μg sequestration agent per 100 LD₅₀ units botulinumtoxin. In a preferred embodiment, the sequestration agent is present inan amount between 13,000 and 50,500 μg sequestration agent per 100 LD₅₀units botulinum toxin. In a more preferred embodiment, the sequestrationagent is present in an amount between 50,500 and 505,000 μgsequestration agent per 100 LD₅₀ units botulinum toxin. In the mostpreferred embodiment, the sequestration agent is formulated asencapsulated microspheres in an amount between 50,500 and 90,500 μgsequestration agent per 100 LD₅₀ units botulinum toxin.

The botulinum toxin of the present compositions may be selected from avariety of strains of Clostridium botulinum. In a preferred embodiment,the compositions of the present invention comprises a botulinum toxinselected from the group consisting of botulinum toxin types A, B, C, D,E, F and G. In a preferred embodiment, the botulinum toxin is botulinumtoxin type A. In a more preferred embodiment, the botulinum toxin isbotulinum toxin type A from the Hall strain of Clostridium botulinum.

In another embodiment, the compositions of the present inventioncomprise a botulinum toxin that consists essentially offractionated-light-chain botulinum toxin. In yet another embodiment, thebotulinum toxin consists essentially of a mixture of hybrid andchain-translocated forms of botulinum toxin. In a further embodiment,the botulinum toxin consists essentially of chimeric forms of botulinumtoxin. Although the present invention may utilize any botulinum toxin,botulinum toxin fragment that retains neurotoxic activity, botulinumtoxin chimeras and hybrids, chemically-modified botulinum toxin, andspecific activities well known to those of ordinary skill in the art, inone embodiment the botulinum toxin is purified to a specific activitygreater than or equal to 20 LD₅₀ units per nanogram botulinum toxin.

The present invention provides compositions of botulinum toxin and asequestration agent wherein the ratio of LD₅₀ units of botulinum toxinto μg sequestration agent is less than or equal to 0.2 for botulinumtoxin type A and is less than or equal to 10 for botulinum toxin type B.

Each composition of the present invention, in addition to comprising abotulinum toxin and a sequestration agent, may further comprise apharmaceutically acceptable carrier and/or zinc and/or a zinc salt. Inone embodiment, the botulinum toxin is noncovalently bound to thesequestration agent. In another embodiment, the botulinum toxin iscovalently bound to the sequestration agent.

The present invention provides compositions of a botulinum toxin and asequestration agent, wherein the sequestration agent is selected fromthe group consisting of: proteins, lipids and carbohydrates. In apreferred embodiment, the sequestration agent is albumin, collagen,epinephrine or hyaluronate. In a more preferred embodiment, thesequestration agent is hyaluronate. In the most preferred embodiment,the sequestration agent is albumin.

The present invention further provides compositions comprising abotulinum toxin and a sequestration agent, wherein the sequestrationagent is an albumin, preferably human serum albumin. Furthermore, in oneembodiment, the albumin of the present compositions is recombinantlyproduced. In one embodiment, the albumin is present in an amount between550 and 5,500 μg albumin per 100 LD₅₀ units botulinum toxin. In afurther embodiment, albumin is present in an amount between 5,500 and13,000 μg albumin per 100 LD₅₀ units botulinum toxin. In a preferredembodiment, albumin is present in an amount between 13,000 and 50,500 μgalbumin per 100 LD₅₀ units botulinum toxin. In a more preferredembodiment, albumin is present in an amount between 50,500 and 505,000μg albumin per 100 LD₅₀ units botulinum toxin. In a most preferredembodiment, albumin is formulated as encapsulated microspheres in anamount between 50,500 and 90,500 μg albumin per 100 LD₅₀ units botulinumtoxin.

In another embodiment, the present invention provides a compositioncomprising botulinum toxin and a sequestration agent, wherein thesequestration agent is present in an amount between 550 and 900,500 μgsequestration agent per 100 LD₅₀ units botulinum toxin, wherein thealbumin may be formulated as a solid albumin particle.

In one embodiment of the present invention, the compositions comprise abotulinum toxin and at least one sequestration agent. In a preferredembodiment, the compositions of the present invention comprising abotulinum toxin and albumin and further comprising one or moreadditional sequestration agents.

The present invention also provides methods of producing localizeddenervation in a subject in need thereof, comprising administering aneffective amount of any of the compositions of the present inventionthat are described herein. In one embodiment, the methods of the presentinvention are used to produce denervation in a subject that suffers froma neuromuscular disease associated with increased muscle tone withinvoluntary movement. In another embodiment, the methods of the presentinvention are used to produce denervation in a subject that suffers froma neuromuscular disease. Preferably, the neuromuscular disease ischaracterized by increased muscle tone and/or involuntary movement,including but not limited to dystonias, spinal cord injury or disease,multiple sclerosis, spasticity, cerebral palsy, stroke, and the like.Preferably, the neuromuscular disease associated with increased muscletone and/or involuntary movement is blepharospasm or torticollis. Morepreferably, the neuromuscular disease associated with increased muscletone with involuntary movement is blepharospasm.

In one embodiment, the present invention provides methods for producingdenervation in a subject suffering from blepharospasm comprisingadministering between 10-200 LD₅₀ units of a composition of the presentinvention, as described herein. In another embodiment, the presentinvention provides methods for producing denervation in a subjectsuffering from torticollis. Preferably, the effective amount of acomposition of the present invention is between 10 and 3000 LD₅₀ units.

In another embodiment, the present invention provides a method oftreating a condition selected from the group consisting of facialwrinkles, rhytides and cosmetic alteration of lip and brow, in a subjectin need thereof, comprising administering an effective amount of acomposition of the present invention, as disclosed herein. Preferably,the effective amount is between 2.5 and 400 LD₅₀ units.

In yet another embodiment, the present invention provides a method oftreating human headache disorders in a subject in need thereof,comprising administering an effective amount of a composition of thepresent invention, as disclosed herein. Preferably, the effective amountis between 5 and 1000 LD₅₀ units.

In a further embodiment, the present invention provides a method oftreating human migraine headache disorders in a subject in need thereof,comprising administering an effective amount of a composition of thepresent invention, as disclosed herein. Preferably, the effective amountis between 5 and 1,000 LD₅₀ units.

The present invention also provides a method of treating humaninflammatory conditions in a subject in need thereof, comprisingadministering an effective amount of a composition of the presentinvention, as disclosed herein. Preferably, the effective amount isbetween 5 and 4,000 LD₅₀ units.

The present invention also provides a method of treating myopathic orneuropathic pain in a subject in need thereof, comprising administeringan effective amount of a composition of the present invention, asdisclosed herein. Preferably, the effective amount is between 5 and4,000 LD₅₀ units.

The present invention also provides a method of treating back pain orarthritic pain in a subject in need thereof, comprising administering aneffective amount of a composition of the present invention, as disclosedherein. Preferably, the effective amount is between 5 and 4,000 LD₅₀units.

In yet another embodiment, the present invention provides a method oftreating gastrointestinal spasm and strictures in a subject in needthereof, comprising administering an effective amount of a compositionof the present invention, as disclosed herein. Preferably, the effectiveamount is between 5 and 4,000 LD₅₀ units.

The present invention provides a method of treating a hyperhyrosissyndrome in a subject in need thereof, comprising administering aneffective amount of a composition of the present invention, as disclosedherein. Preferably, the effective amount is between 5 and 4,000 LD₅₀units.

The present invention also provides a method of producing thecompositions described herein. In one embodiment, the method comprisesmixing a sequestration agent with botulinum toxin. In anotherembodiment, the method comprises freeze drying or flash drying asequestration agent with botulinum toxin. Preferably, the botulinumtoxin and the sequestration agent are in a weight to weight ratio whichexceeds 100 μg sequestration agent to 1 ng of botulinum toxin.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application filed contains at least one drawing executedin color. Copies of this patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 depicts the denervation potency of two botulinum toxinpreparations as determined by muscle fiber variability.

FIG. 2 depicts the denervation potency of two botulinum toxinpreparations as determined by histochemical methods(acetylcholinesterase staining).

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a method and composition to enhance theclinical effectiveness of botulinum-toxin preparation for clinical useby means of increasing sequestration of botulinum neurotoxin moleculesin the region targeted for therapy through the use of a sequestrationagent or “molecular anchor”. Enhanced sequestration using higherconcentration of macromolecules such as proteins (e.g., albumin,collagen and the like), and/or lipids and/or polysaccharides (e.g.,hyaluronate, and the like) can be useful to provide a molecular anchorto neurotoxin molecules preventing diffusion away from the injectionpoint, causing maximal saturation of botulinum neurotoxin receptors,thereby achieving greater efficacy with the amount of neurotoxin used toachieve desired clinical effects. The sequestration agent enhancescontainment of regional denervation, and enhances clinical outcomes. Theincreased sequestration allows for better delivery to nerve ending, withenhanced uptake and augmentation of denervative and other biologiceffects. The invention requires a sequestration agent added to aformulation of neurotoxin which binds to the neurotoxin, preventsdissemination of the neurotoxin and demonstrates improvement in clinicalresponse in patients who were previously treated without the carriermolecule at preferred concentrations. The sequestration agent may be anexisting excipient at significantly higher concentrations thanpreviously used (such as human serum albumin), or a material that hasnot been previously used to stabilize botulinum toxin (such as sodiumhyaluronate). The sequestration agent must bind to the botulinum toxinmolecule and prevents its diffusion so that the neurotoxin may reactwith the nerve-terminal ending or any neural structure so thateffectiveness of the therapy is improved.

A. DEFINITIONS

As used herein, “Botulinum toxin” means a protein toxin isolated fromstrains of Clostridium botulinum, including mixtures of its proteincomplexes, toxoid and/or other clostridial proteins. “Botulinum toxin”includes all of the various immunotypes such as A, B, C₁, C₂, C₃, D, E,F and G.

As used herein, “an effective amount” is an amount sufficient to producea therapeutic response. An effective amount may be determined with doseescalation studies in open-labeled clinical trials or bin studies withblinded trials.

As used herein, “increased clinical potency” means that fewer LD₅₀ Unitsof a pharmaceutical formulation comprising a botulinum toxin and asequestration agent (as described herein) are necessary for “aneffective amount” of a botulinum toxin for the treatment of glabellarlines than a botulinum toxin preparation without an added sequestrationagent.

As used herein “neuromuscular diseases” refer to any disease adverselyaffecting both nervous elements (brain, spinal cord, peripheral nerve)or muscle (striated or smooth muscle), including but not limited toinvoluntary movement disorders, dystonias, spinal cord injury ordisease, multiple sclerosis, and spasticity from cerebral palsy, stroke,or other cause.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition, compound, or solvent with which an activeingredient may be combined and which, following the combination, can beused to administer the active ingredient to a subject. As used herein,“pharmaceutically acceptable carrier” includes, but is not limited to,one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; preservatives;physiologically degradable compositions such as gelatin; aqueousvehicles and solvents; oily vehicles and solvents; suspending agents;dispersing or wetting agents; emulsifying agents, demulcents; buffers;salts; thickening agents; fillers; antioxidants; stabilizing agents; andpharmaceutically acceptable polymeric or hydrophobic materials and otheringredients known in the art and described, for example in Genaro, ed.,1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., which is incorporated herein by reference.

As used herein, “sequestration agent” means an agent that enhanceslocalization and/or retention of the botulinum toxin to the site ofadministration.

B. BOTULINUM TOXIN

Botulinum toxin type A is the most lethal natural biological agent knownto man. Seven immunologically distinct botulinum neurotoxin serotypeshave been characterized—A, B, C₁, D, E, F and G. Each botulinum toxinserotype is distinguished by neutralization with type-specificantibodies. The different serotypes of botulinum toxin vary in theanimal species that they affect and in the severity and duration of theparalysis they evoke.

Since its introduction as a therapeutic agent, the pharmaceuticalmeasurement of the denervating or biologic activity of botulinum toxinhas been the LD₅₀ Unit (LD₅₀ Unit and Unit are used interchangeablyherein) determined by using 18-22 gram Swiss-Webster mice, quantitatedstatistically by injecting cohorts of mice at different dilutions fromthe purified botulinum neurotoxin protein and its protein complexes.This measurement has the advantage of a clear endpoint (living or deadmouse), however the LD₅₀ unit does not predict clinical behavior ofvarious botulinum toxin formulations when compared in clinical studies.For instance, one preparation of type B botulinum toxin (MYOBLOC®)requires 5,000-15,000 LD₅₀ units to treat torticollis whereas anotherpreparation of botulinum toxin Type A (BOTOX®) requires only 100-300LD₅₀ units. Similarly, the LD₅₀ unit has failed to distinguishdifferences in therapeutic behavior of different sources of the samebotulinum toxin immunotype. For instance, approximately 50-300 units ofBOTOX® is required to treat blepharospasm and cervical dystonia comparedto 200-1200 units of DYSPORT®, another preparation of botulinum type Atoxin. Table 1 illustrates the varying doses for different diseases.

TABLE 1 Dosing comparisons between various pharmaceutical formulationsof botulinum toxin. Essential Formulation Blepharospasm TorticollisBOTOX ® 50 U¹ 200 U DYSPORT ® 200 U 600-1,200 U MYOBLOC ® 3,000-5,000 U10,000-15,000 U ¹Units (U) are LD₅₀ units determined using 20-30 gSwiss-Webster mice, as described herein.

Toxins of the different C. botulinum serotypes are produced in cultureas aggregates of neurotoxin and other non-toxic proteins non-covalentlyassociated into a polypeptide complex. (Schantz (1964) Purification andcharacterization of C. botulinum toxins, In Botulism. Proceedings of asymposium. K. Lewis and K. Cassel, Jr. (eds.), U.S. Department ofHealth, Education, and Welfare, Public Health Service, Cincinnati, pp.91-104; Sugii et al. (1975) Infect. Immun. 12: 1262-1270; Kozaki et al.,(1974) Jpn. J. Med. Sci. Biol. 28: 70-72; Miyazaki et al. (1977) Infect.Immun. 17: 395-401; Kitamura et al. (1969) J. Bacteriol. 98: 1173-1178;Ohishi et al. (1974) Appl. Environ. Microbiol. 28: 923-928; Yang et al.(1975) Appl. Microbiol. 29: 598-603). Toxin complexes are described as Mfor medium, L for large and LL for very large. These toxin complexesvary in size from about 900 kD for type A LL toxin complex to about 300kD for the type B M complex and type E complex, to 235 kD for type F Mcomplex. The Hall strain of type A Clostridium botulinum is preferablyused for the production of type A neurotoxin. (Goodnough et al. (1992)Appl. Environ. Microbiol. 58(10): 3426-3428); Goodnough and Johnson(1994) ACS Symposium Series No. 567, J. Cleland and R. Langer (eds); Tseet al. (1982) Eur. J. Biochem. 122: 493-500). Botulinum neurotoxin maybe prepared by culturing Clostridium botulinum, harvesting, solubilizingand purifying using standardized methods that ensure quality andsterility. (Schantz and Johnson (1992) Microbiol. Rev. 56: 80-99;(Goodnough et al. (1992) Appl. Environ. Microbiol. 58(10): 3426-3428);Goodnough and Johnson (1994) ACS Symposium Series No. 567, J. Clelandand R. Langer (eds); Tse et al. (1982) Eur. J. Biochem. 122: 493-500).incorporated herein by reference in its entirety).

C. ALBUMIN

Endogenous human serum albumin binds native circulating molecules, suchas free fatty acids, bilrubin, hormones and zinc. Additionally,circulating human albumin can bind with many pharmaceutical agents whichcan influence potency, complication rate, clearance, and otherpharmacodynamic properties of these agents. Examples includesalicylates, sulfisoxazoles, warfarin, phenylbutazone, digitoxin,phenyloin, oxacillin, benyzlpenicillin, lasix, indomethacin, diazepam,and quinidine among others. Peptides and proteins also are known to bindhuman serum albumin. Peptide hormones such as gastrin, corticotropin,melatonin are also known to bind human serum albumin.

Several binding sites have been identified and binding has been thoughtto be non-covalent. Additionally, albumin can non-covalently bindcations that serve as cofactors for enzymatic reactivity of portions ofthe botulinum toxin polypeptide complex. Specifically, zinc is acofactor for the endopeptidase activity of the botulinum toxin lightchain which enters the target cells after heavy chain binding to thecell surface protein receptors. Higher quantities of zinc bound toalbumin enhance endopeptidase activity. Zinc binding to albumin is dosedependent. Saturation of zinc binding on albumin enhances thedenervating effect of botulinum toxin.

Albumin, because of larger atomic mass and other protein properties, isphysiologically cleared from the injection area by lymph vesselabsorption, not blood vessel absorption), a process which is much slowerthan removal of smaller molecular species. The relevance of albumin tobotulinum toxin pharmaceuticals depends on its role in maintainingbiologic activity by promoting nerve and other receptor contact andpreventing wash out from free neurotoxin release at injection points.DYSPORT®, with its lower albumin concentration, offers lesssequestration for the neurotoxin complex, and subsequently, afterinjected, diffusion away from the targeted anatomic area results. Theclinical effect is a greater regional diffusion and chemodenervationover greater area, which results in increased complications (ptosis,Dyspahgia see Table 2). In order to compensate for this behavior,clinicians have given four to five time as much neurotoxin to achievethe same degree of biologic activity as formulations such as BOTOX® havehigher albumin concentrations. With less potent immunotypes such asbotulinum toxin type B (MYOBLOC®), larger dose are needed to achieve thesame regional bioeffect, thereby further increasing diffusion andcomplication rates (see Table 2). The lower potency observed forimmunotypes are thought to be related to poor receptor binding orbinding to alternative less efficient receptor sites. Administration ofmore botulinum toxin (higher protein load), in addition to increaseddiffusion, also results in higher immunity rates after repeatedinjections. (Borodic et al. (1996) Botulinum Toxin, Immunology andProblems with Available Materials. Neurology 46: 26-29).

MYOBLOC® is formulated at an acidic pH<6.0 which provides for increasedstability and stability of the liquid formulation at room temperature.Unfortunately, the acidic pH has an adverse side effect on the structureand probably tissue carrying properties of the human serum albumin inthis formulation. The isomerization, tertiary structure and physicalproperties of albumin can vary considerably at various pH. (see Peters(1996) All about Albumin. Academic Press, New York; incorporated hereinby reference in its entirety). Alterations in physical properties (viachanges in binding of botulinum toxin and the dynamics of botulinumtoxin molecular release in tissues) may contribute to differences indose requirements comparing BOTOX® and MYOBLOC® in clinical practice.

Although other proteins (e.g. gelatin, lactalbumin, lysozyme), lipidsand carbohydrates may serve as effective sequestration agents, albumin,including encapsulated albumin and solid microspheres is the preferredprotein sequestration agent, in part, because of its low immunogenicity.Other proteins, polysaccharides, lipids, polymers, gels and hydrogelsthat are potentially suitable as sequestration agents are disclosed inU.S. Pat. No. 4,861,627, which is incorporated herein by reference inits entirety. Methods of using and making protein microspheres,including albumin microspheres, are disclosed in U.S. Pat. Nos.6,620,617; 6,210,707; 6,100,306; and 5,069,936 which are eachincorporated herein by reference in their entirety.

D. SEQUESTRATION

The concept of sequestration has been used by the inventor to explainaltered lidocaine toxicity when periocular injections are given in theabsence of Wydase. (Troll et al. (1999) Diplopia after cataract surgeryusing 4% lidocaine in the absence of Wydase™. Clin Anesth. 11(7):615-6). Diffusion, in the absence of Wydase, of injectable lidocaine inthis circumstance causes toxicity of myofibrils of the extra-ocularmuscles with contraction scarring and damage to extra-ocular movement.The lidocaine example indicates how sequestration from dynamic diffusionof an injectable drug can be important to the drug's basic pharmacology.

There has, however, never been a suggestion or recommendation thatalbumin can alter regional denervation potency or enhance clinicaleffects or be used to treat patients not responding to BOTOX®, DYSPORT®or MYOBLOC®. The present invention provides compositions and methodsthat enhance the clinical effectiveness of botulinum toxinpharmaceuticals.

As pointed out in the potency section above, sequestration—the regionalcontainment of chemodenervation—is one of the most important propertiesof the formulations of the present invention. Minimizing diffusionenhances potency, reduces diffusion associated complications, andreducing botulinum toxin antigenicity because lower doses are necessaryto achieve a therapeutic effect. Preparations which require higherdosing, that is administration of an increased protein load, areassociated with higher rates of immunity (comparing 79-11 originalOculinum Batch to current BOTOX® Batch, MYOBLOC® compared to BOTOX®).Enhanced sequestration allows for lower protein load, less diffusion,and enhanced biologic effect within the region targeted for treatment.The utility of this improved composition is demonstrated by itstherapeutic effectiveness when conventional formulations (e.g., BOTOX®,MYOBLOC®) currently in use have failed or given suboptimal results.

E. DOSING OF HIGH-POTENCY BOTULINUM TOXIN FORMULATIONS

Isolated type A toxin complex has a specific toxicity of about 20-40LD₅₀/ng in 18-22 g Swiss-Webster mice. Specific toxicities of other C.botulinum toxin complexes are 40-50 LD₅₀/ng for the type B M complex;10-20 LD₅₀/ng for the type C₁ M complex; 70-80 LD₅₀/ng for the type D Mcomplex; 10 LD₅₀/ng for the type E M complex; 20-30 LD₅₀/ng for the typeF M complex (Sugiyama (1980) Microbiol. Rev. 44: 419-448); and 80-90LD₅₀/ng for the type G complex (Schiavo et al. (1994) J. Biol. Chem.269: 20213-20216).

Type A neurotoxin produced by C. botulinum is present as part of acomplex of at least seven different noncovalently bound proteins. Highquality type A toxin complex has a specific neurotoxicity of 30 LD₅₀Units/nanogram. The purified neurotoxin, that is the neurotoxin that hasbeen chromatographically separated from the other proteins of the toxincomplex, has a specific toxicity of between about 100 and 250 LD₅₀Units/nanogram. Clinically, a unit (U) is considered to be 1 LD₅₀. Oneunit of botulinum neurotoxin is defined as the LD₅₀ upon intraperitonealinjection into female Swiss Webster mice weighing 18-22 grams each, orabout 50 picograms a purified botulinum neurotoxin (essentially free ofbotulinum toxin complex proteins). (Schantz and Kautter (1978)Association of Official and Analytical Chemistry 61: 96; See also, U.S.Pat. No. 5,512,547).

The current commercial type A botulinum toxin (BOTOX®) is produced bycombining up to 500 ng/ml of type A toxin complex in 5.0 mg/ml humanserum albumin (HSA) with 9.0 mg/ml sodium chloride at a pH of 7.3. Thepre-lyophilization fluid is reduced to 0.1 ml. After dissolution, 0.1 mlis dried to obtain 100±30 Units of toxin, 0.5 mg of HSA, and 0.9 mg ofsodium chloride per vial. This product has a saline concentration of0.9% when reconstituted in 1.0 ml of water. The original commercialformulation of BOTOX®, which employs the toxin complex, had a specificneurotoxicity of about 2.5 U/ng after drying (Allergan, Inc. of Irvine,Calif.). The considerable loss (up to 90%) of activity during dryingcauses the formation of inactive toxin (toxoid) that can induce antibodyformation. More recently, improvements have been made to raise thespecific toxicity of BOTOX® to 18-20 U/ng neurotoxin.

Compositions of botulinum toxin that require a lower effective amount totreat particular conditions are particularly desirable, because theadministration of botulinum toxin has been associated with thedevelopment of immunologic resistance. Consequently, this complicationrequires increased dosing (higher LD₅₀ units) to achieve atherapeutically-effective amount of the botulinum toxin. Ultimately,immunity renders the use of botulinum toxin ineffective.

A composition of Hall-strain-derived botulinum toxin was formulated witha specific activity of 20 LD₅₀ units/ng toxin and 900 μg human serumalbumin to 100 LD_(50 units of botulinum toxin ()0.11 LD₅₀ unit/μgalbumin)(US FDA IND 4891). The indication for therapy for this newformulation was aberrant regeneration of the facial nerve withinvoluntary synkinetic blepharospasm. The study was conducted usingbetween 5 and 15 LD₅₀ units of botulinum type A toxin formulated withthe increased amount of albumin to LD₅₀ content.

TABLE 5 Reduction in effective amount of botulinum toxin usinghigh-albumin botulinum toxin compositions. Open-Lable 15 patients 100%demonstrated No ptosis Trials each receiving 5-15 decreased involuntarycomplication LD₅₀ units movement Double-Blind Placebo 30 patients (ratio1:1 1. Degree of involuntary No ptosis Controlled Trialstreatment/control) each movements significantly complications receiving15 LD₅₀ units better than controls. 2. Subjective parameterssignificantly better than controls

Prior literature has indicated that existing BOTOX® preparations require20 LD₅₀ units to achieve favorable results for this indication. (Borodicet al. (1993) Botulinum Toxin for aberrant facial nerve regeneration.Dose response relationships. Plastic and Reconstructive Surgery, (91)₆:1042-1045. 1993). Furthermore, there has been a 20% incidence of ptosis(a diffusion complication) associated with the use of botulinum toxinfor involuntary blepharospasm, based on a 100 patient study on BOTOX®for the treatment of blepharospasm and using comparable LD₅₀ doses (seenew batch approval study from Allergan Pharmaceuticals, 1998;incorporated herein by reference in its entirety). Comparing theincidence of this complication in the high-albumin study shown abovewith the BOTOX® equivalency study (19/99, compared to 0/30, P<0.01, ChiSquare), it appears that the high-albumin type A botulinum toxincomposition required fewer LD₅₀ units to achieve acceptable therapeuticresults (reduction in effective amount of toxin) and was associated withlimited diffusion into the orbit which frequently results in ptosis. Thedecreased incidence of this complication indicated sequestration of theeffects of botulinum toxin was enhanced by the higher albumin content.

In the clinical setting, botulinum neurotoxin type A (BOTOX®) is used atdoses that vary depending on the clinical indication and the size andtype of the muscle being treated. The following dosing ranges exemplifythe clinical use of BOTOX®: (1) about 75-125 Units of BOTOX® perintramuscular injection (multiple muscles) to treat cervical dystonia(200-300 Units per injection cycle); (2) 5-10 Units of BOTOX® perintramuscular injection to treat glabellar lines (brow furrows)(20-50Units per injection cycle)(5 units injected intramuscularly into theprocerus muscle and 10 Units injected intramuscularly into eachcorrugator supercilii muscle, and frontalis and orbicularis muscles);(3) about 30-80 Units of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle; (4) about 20-40 Units per muscleof intramuscularly injected BOTOX® to treat blepharospasm by injectingthe lateral pre-tarsal orbicularis oculi muscle of the upper lid and thelateral pre-tarsal orbicularis oculi of the lower lid; (5) to treatstrabismus, extraocular muscles have been injected intramuscularly withbetween about 2.5-10 Units of BOTOX®, the amount injected varying basedupon both the size of the muscle to be injected and the extent of muscleparalysis desired (i.e. amount of diopter correction desired); and (6)to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows: (a) flexor digitorum profundus: 7.5-30 Units; (b) flexordigitorum sublimus: 7.5-30 Units; (c) flexor carpii ulnaris: 10-40Units; (d) flexor carpii radialis: 15-60 Units; (e) biceps brachii:50-200 Units. (See, U.S. Pat. No. 6,358,926 (col. 5, lines 18-48); U.S.Patent Publication No. 20020197278).

F. POTENCY

The potency of a particular botulinum toxin preparation or formulationmay be determined clinically or in animal models of muscle denervation.Clinically, a first botulinum toxin preparation exhibits greater potencythan a second preparation when fewer LD₅₀ Units of the first preparationare required to achieve a desired therapeutic effect.

In animal models, the potency of a botulinum toxin preparation may bedetermined by measuring the extent of denervation produced when apreparation is administered to a muscle. Post mortem sectioning ofrabbit muscle about a site of toxin injection, demonstrates thatbotulinum toxin produces a gradient of denervation similar to thatobserved in mouse muscle (Duchen (1970) J. Neurol. Neurosurg. 33:40-54;J. Physiol. (Lond) (1969) 204:17-18). The extent of this denervationgradient (a measure of the spread of a given dose of the toxin) is ameasure of potency. Animal models for muscle denervation are disclosedand described in U.S. Pat. No. 5,298,019, which is incorporated hereinby reference in its entirety.

The longissimus dorsi muscle of New Zealand white rabbits is thepreferred animal model for determining the denervating potency of abotulinum toxin preparation. Denervation may be assessed by anyavailable analytical method. For example, denervation may be determinedat various distances from the injection site by post mortem sectioningof the treated muscle and staining for acetylcholinesterase activity.Techniques for acetylcholinesterase-activity staining are described byKarnovsky (See Woolf and Coers, The Innervation of Muscle, CharlesThomas Pub, Springfield, Ill., 1959, which is incorporated by referenceherein in its entirety). Inhibition of acetylcholine release may also bemeasured by single-fiber electromyography (See, for example, Sanders etal. (1985) Botulinum Toxin for Blepharospasm, Single Fiber EMG Studies,Neurology 35: 271-272). Labeled binding proteins, including polyclonalor monoclonal antibodies, may also be used to detectacetylcholinesterase, acetylcholine receptors, and acetylcholinesteraseactivity. Binding proteins may be labeled using, for example,fluorescein or other fluorescent moieties, colloidal metallic particles,other remotely-detectable substances, and the like. Antibodies can beproduced, using known techniques, to acetylcholine receptors or toacetylcholinesterase, both of which can serve as a marker for effectivedenervation, or to epitopes which are newly exposed, or which remainafter binding of the toxin to the receptor on the presynaptic motor endplate. Other stains such as hematoxylin, eosin, masson trichrome, andthe like may also be used.

G. NEUROMUSCULAR DISORDERS

The botulinum toxin formulations of the present invention may be used totreat a variety of neuromuscular disorders that are characterized byinvoluntary muscle contractions and/or spasms. These neuromusculardisorders include, but are not limited to dystonias, including cervicaldystonia (spasmodic torticollis), spasmodic dysphonia, hemifacial spasm,blepharospasm, bruxism, and spasticity caused by cerebral palsy, stroke,and the like.

Cervical dystonia or spasmodic torticollis is a focal dystoniacharacterized by neck muscles contracting involuntarily, causingabnormal movements and posture of the head and neck. The abnormalmovements and spasms may occur in any direction. Contractions producingforward movements are frequently referred to as anterocollis, whereasspasm that produce backwards or sideways movements are referred to asretrocollis and torticollis, respectively. The movements may besustained or sporadic. Sustained contractions produce abnormal head andneck posture, whereas periodic spasm produce jerky head movements. Thespasms and muscle contractions that produce cervical dystonia are alsoassociated with considerable neck pain and discomfort.

The cause of cervical dystonia is unknown, but is believed to beassociated with defects in the basal ganglia which control movement.Although a dopamine deficiency or imbalance may be the underlyingchemical basis for the disorder, the exact etiology of cervical dystoniaremains unknown. Cervical dystonia may be diagnosed through a medicalhistory, physical and neurological examination. Currently, there is nolaboratory or clinical test to confirm a diagnosis of blepharospasm.

Cervical dystonias usually increase in severity, reaching a plateau andremaining stable within five years after onset. This form is unlikely tospread or become generalized dystonia, though patients with generalizeddystonia may also have cervical dystonia. Occasionally, there may beassociated focal dystonia. Cervical dystonia should not be confused withother conditions which cause a twisted neck such as local orthopedic,congenital problems of the neck, ophthalmologic conditions where thehead tilts to compensate for double vision. It is sometimes misdiagnosedas stiff neck, arthritis, or wry neck.

Botulinum toxin injections are the primary and most effective form oftreatment for cervical dystonia. Injections are made directly into theaffected neck muscles. A crucial element to successful botulinum toxininjections is that the appropriate muscles are injected. For example,the muscles most commonly involved in cervical dystonia include thesplenius capitis, the levator scapulae, upper trapezius,sternocleidomastoid, anterior, middle and posterior scalene. TheDystonia Medical Research Foundation, for example, recommends low-dose(about 150 U BOTOX®) administration of botulinum toxin to avoidimmunity. Single and especially chronic dosing with greater than 200 UBOTOX® greatly increases the risk of inducing the production ofneutralizing antibodies and resistance to the toxin.

H. NEUROSENSORY DISORDERS (PAIN)

The botulinum toxin formulations of the present invention may also beused to treat a variety of sensory disorders such as pain syndromes,including myofascial pain, migraine, tension headaches, post-operativewound pain, nerve compression, neuralgias, trigeminal neuralgia, painassociated with cervical dystonia and other dystonias, neuropathy, andsinusitis-related facial pain.

Sinus-related headaches are distinctly different from migraine headache,myofascial headaches, and headaches associated with bruxism, temporalmandibular joint syndrome (TMJ) and temporal mandibular muscledysfunction (TMD), trigeminal neuralgia, tooth related facial pain, painassociated with elevated intraocular pressure, or internal ocularinflammation. Sinus headaches are associated with pressure, orirritating processes within the sinus cavities, sometimes associatedwith inflammation and impaired flow of mucous secretion. At some pointin the diagnostic workup, excessive signs of inflammation within thesinus or nasal cavity, or edema within the sinus or nasal cavity isdemonstrated on exam or via radiographic methods. The present inventorshave discovered that botulinum toxin relieves the headache and facialpain associated with sinusitis.

The present invention provides methods of treating headache and facialpain associated with acute recurrent or chronic sinusitis in a subjectin need thereof, comprising the step of administering a therapeuticallyeffective amount of a composition comprising botulinum toxin to thenasal mucosa or to the subcutaneous structures overlying the sinuses,wherein the administration of the composition reduces the headache andfacial pain associated with acute recurrent or chronic sinusitis. In apreferred embodiment, the sinuses are one or more of the sinusesselected from the group consisting of: ethmoid; maxillary; mastoid;frontal; and sphenoid. Preferably, the subcutaneous structures overlyingthe sinuses lie within one or more of the areas selected from the groupconsisting of: forehead; malar; temporal; post auricular; and lip.

Botulinum toxin may be administered to the nasal mucosa or to thesubcutaneous structures overlying the sinuses by any number of methods.Preferably, the composition comprising botulinum toxin is administeredby injection at one or more injection sites. More preferably, thecomposition comprising botulinum toxin is administered to the cutaneousprojections of the trigeminal nerve innervating the sinus.

In one embodiment of the present invention, a subject is treated byadministration of a composition comprising botulinum toxin, wherein thesubject, prior to the onset of facial pain or headache, exhibitssymptoms or history of sinus rhinorrhea (nasal hypersecretion) andpurulent nasal discharge.

Sinusitis is defined as any inflammatory pathology involving theethmoid, maxillary, frontal, or sphenoid sinuses. It is generallyaccepted that the cause of pain occurring with acute sinusitis involvesinfiltration of sinus mucosa with inflammatory cells, as well asincreased pressure within the sinuses. What is generally notappreciated, and is herein disclosed, is that sinusitis can causesensitization of the trigeminal nerve in cutaneous and subcutaneoustissues overlaying the sinus structures. When sensitization of sensorynerves occurs from repeated bouts of sinusitis, the patient canexperience a chronic facial pain syndrome or headache. The mechanism bywhich sensory nerves become up-regulated or sensitized still is notclear. Nerve sensitization is provoked by alterations in the afferentfirst-order-sensory nervous system, such that thresholds are lowered tothe perception of pain (hyperalgesia) and central second-order orhigher-neuronal alterations can occur, resulting in an exaggeratedresponse and interpretation of sensory stimuli (central sensitization).This process has been experimentally associated with increasedexpression and/or responsiveness of NMDA receptors on membranes ofnociceptors and possible alterations in transcription and translation ofproteins within the nerve cell. The trigeminal ganglia represent a verylarge collection of afferent sensory neurons, which send projects notonly into cutaneous regions of the head, but also internally intoosseous sinus structures, and mucous membranes of the nasal and sinuscavities.

The arborization pattern of afferent sensory nerve distribution isextensive, but reactivity within any region of the afferent sensorynerve distribution has the capability of altering the genetic andcellular-protein expression of the sensory nerve cell body within theganglion. The process of changing cell physiology has been variouslycoined neuroplasticity or sensitization. Alterations can be in the formof increased expression of nerve cell receptors, such as AMPA and NMDAreceptors, modulation of effectors of inflammation, alteration ofcellular responses from blood-vessel neural regulation via nitric oxide,substance P, histamine, CRGP, prostaglandins, other known cellularautocoids, and not yet defined autocoids and neuropeptides. Themechanism for sensitization of human nerve cells is still not wellunderstood, and invoking inflammatory mediators, neurogenic inflammatoryautocoids, and transcriptional and phenotypic changes of nociceptors andsensory neurons as the only mechanisms for nerve sensitization is notnecessary to elicit responses from therapeutic botulinum toxin for thisindication. Sensitization in the periphery is thought to occur followinga sufficient or prolonged exposure to inflammatory substances, causingaltered physiology, possible conformational changes of certainbiochemical receptors, responsiveness, and lowered thresholds fornociceptor and sensory nerve depolarization.

Sinus pain usually begins in the mid facial region over the maxillarysinus and can radiate to temporal regions, ocular regions, vertex, andover the forehead. At times, referred pain can project into theposterior cervical region or peri-auricular areas. Generalized headachescan occur. The trigeminal nucleus is somatotropically well organized,and from the brain stem area, directly extends and connects anatomicallyto the upper-cervical areas of the dorsal horn of the spinal cord. Inaddition, there are interneuronal connections between the trigeminalnucleus and other cranial nerve nuclei, the autonomic nervous system,the reticular activating system, and other descending and ascendingpathways. This interconnecting system has been described as thetrigeminal sensory complex. Since there are many more peripheral uppercervical and trigeminal sensory nerves synapsing on fewer centralnerves, this has been described as convergence and projection. This canexplain the referral patterns of head and neck pains, and the therapiesemployed in one area of the head and neck to affect an outcome on aanother area of the head and neck with shared and referred sensorypathways.

Distinct differences in headache diagnosis have been formulated atinternational conventions and remain the basis for both general andresearch practice. For migraine headaches, the presence of episodicheadaches lasting 4-48 hrs, associated with light sensitivity(photophobia), sound sensitivity (phonophobia), nausea or vomiting, painof a throbbing or pulsating quality, and more often unilateral thanbilateral location of headache. Cluster headaches can be associated withsome basal transient nasal congestion but occur over a distinct timeperiod (cluster period) and are not associated with any persistent sinusabnormalities on MRI or computerized tomography. Myofascial and tensionheadaches often have a cap-like squeezing pain across and around the topof the head, often associated with a cervical musculoskeletal painlocation, frequently associated with trigger points, and sometimesassociated with decreased jaw motility and bruxism if the masseter andtemporalis muscles are involved. Ocular-related headaches are associatedwith increased intra-ocular pressure or signs of intra-ocularinflammation on slit lamp microscopic exam or measured refractive error.Dental-related headaches are associated with findings on dentalexamination and radiographs. Trigeminal neuralgia is usually limited toone or two dermatomes and is sharp and stabbing in quality, with a rapid“on-off” episodic pattern sometimes associated with stimulation oftrigger points.

Chronic-sinusitis-related headache and facial pain can linger for manymonths to years after an acute or subacute bout of sinus disease or boutof repeated acute sinus headaches. Often, the patient complains ofcontinued pain when radiologic imaging studies, such as computerizedtomography and magnetic resonance imaging fail to show any persistingsigns of inflammation such as mucosal thickening or fluid accumulation.Often out of desperation, the surgeon performs decompressive surgery viaendoscopes or direct approaches (Culdwell luc, external ethmoidectomy)with poor results with respect to the chronic pain. The aboveobservation explains a very common clinical phenomenon associated withchronic facial pain and headache caused by sinusitis. The reason for thepersisting pain despite the absence of active sinus findings isperipheral sensory nerve upregulation or sensitization. Direct treatmentof sinus-related headache by botulinum toxin injected into thesubcutaneous region to down-regulate sensory nerves is therapeutic.

The convention in treating sinus-related headaches involvesdecongestants to augment mucous clearance and drainage from sinuscavities, antibiotics to treat bacterial infection, anti-inflammatorymedication (e.g. corticosteroids), and surgical decompression.Conventional analgesics such as aspirin and acetaminophen may be used.The present inventors have made the unexpected discovery thatadministration of botulinum toxin over the surface dermatomes containingthe sensory branches corresponding to the neurons projecting into thesinus cavity effectively treats facial and headache pain associated withsinusitis.

A convention held in 1985 by the International Headache Society (I.H.S.)put forth an exhaustive classification of distinct headache syndromes.Experts in the headache therapeutic field formulated thisclassification, and such experts explicitly agreed on the importance ofheadache distinction both for practice and research. The reasons fordistinctions are to promote better communication among practitioners andto provide more exacting therapy for specific headache syndromes. Forinstance, procedures used to treat trigeminal neuralgia, such asglycerol injections, gamma knife application, and microvasculardecompression at the level of the brainstem are not effective for thetreatment of recurrent sinus headache. Tryptin-related pharmaceuticals(e.g. Imitrex™, Zomig™)) would be ineffective for the treatment of sinusheadache and laser iridectomy for the treatment of narrow angle glaucomawould be ineffective for the treatment of migraine. Cluster headacheneeds to be distinguished from migraine. Hence, one skilled in the artof treatment of pain would require specific and professionallyacceptable diagnosis in order to recommend reasonable therapy or toconduct clinical trials with potentially effective new therapies. Theconvention held in 1985 and subsequently published in Cephalgia (1988Vol 8 (supplement 7), 1-96) has served as a benchmark for diagnosis andclassification of human headaches (nosology) for the past 15 years.

In order for the physician to function and recommend therapeuticinteraction with patients suffering from pain, classification withdiagnostic criteria of an affliction must be determined. Classificationof disease must be operationally specified with quantitative parametersand not just descriptive. The International Headache Society (I.H.S.)formed a committee in 1995 which lead to the first adopted internationalheadache classification, which in turn permitted uniform operationalcriteria for diagnosis. The I.H.S. is internationally accepted and hasbeen incorporated into the World Health Organization (W.H.O.)classification of disease. This classification has been translated intomultiple languages and competes with no other classification system (seeJes Olesen Classification of Headache in Chapter 2, The Headaches,2^(nd) Edition, Lippincott, Williams and Wilkins ed Olesen, Hansen,Walsh, Philadelphia, 1999).

In the classification system, headaches in category 1-4 are primaryheadache disorders with no associated anatomic pathologic process.Groups 5-11 are headaches and cervical pain associated with some otherdemonstrable disease process (trauma, vascular disease, increasedintracranial pressure, withdrawal from substances, systemic infection,metabolic disorder, eye, ear, nose, and throat disease, or dentaldisease. Group 12 relates to cranial neuralgias.

I. INFLAMMATION

Inflammation is a normal response to tissue damage. Inflammation isoften characterized by edema, erythema and pain. Acute inflammation maycaused by a variety of injury, including physical and chemical injuryand tissue damage caused by microorganisms and other agents. Theinflammatory response consist of changes in blood flow, increasedpermeability of blood vessels and the escape of cells from the bloodinto the tissues.

Acute inflammation is short-lasting, lasting only a few days. Chronicinflammation is characterized by a longer duration. Examples of acuteinflammation include hives, swelling, itching and pain associated withinsect bites, burns or exposure to a chemical agent or allergen.Inflammatory conditions may also affect internal organs such as thelungs, gastro-intestinal tract, heart, kidneys and the like.

Disease known to be inflammation driven in etiology include rheumatoidarthritis, inflammatory bowel disease, Crohn's Disease, interstitialcystitis, eczema, hay fever, inclusion arthritis, myositis, postsurgical inflammatory states, reflex sympathetic dystrophy, arteritis,nephritis, scleroderma, asthma, prostatitis, sarcoidosis, bacterialinfections, seborrhea, acne, osteomyleotitis, wound healing sites,systemic lupus erythematosis, Stevens Johnson syndrome, cutaneous anddeep burns, myofascial pain syndromes, osteoarthritis, conjunctitis,blepharitis, uveitis, sialoadenitis, gastritis, tendonitis, keratitis,and post traumatic tissue damage, and the like.

Botulinum toxin in doses lower than that necessary to treat regionalmovement disorders has been shown to reduce inflammation and adversesensory experiences associated with the inflammatory response. Theseobservations are explained by the fact that it has been found that lowdosages of the subject chemodenervative agent reduces histamine releasesand releases of other preformed mediators associated with mast celldegranulation. The anti-inflammatory activity is observed at low dosesin animal models for ocular surface disease that are well noted forhistamine release and release of other preformed mediators associatedwith mast cell degranulation and rapid inflammatory response.Accordingly, botulinum toxin blocks edema, erythema, abnormal sensoryexperiences, and heat transfer that occur rapidly over a predefinedregion.

The anti-inflammatory action of botulinum toxin is explained by theresultant blockage of mast and nerve cell release of histamine and otherpreformed mediators which result in vascular dilation, increasedpermeability, altered sensory experience, edema and erythema—thehallmarks of the rapid-phase inflammatory response. It will beappreciated that mast cells are known to contain a number of substancesimportant to inflammatory responses in hypersensitivity reactions, andsubstantially participate in more generalized inflammatory reactions.The mast cell is abundantly found in pathologic tissue specimens inpatients with rheumatoid arthritis, inflammatory bowel disease, certainforms of ocular uveitis, eczema, and asthma.

Mast cell activation has been associated with the production of bothpreformed mediators such as histamine, newly formed mediators such asleukotrienes and prostaglandins, cytokines, including interleukin-5,interleukin-8, kininogenase, and platelet activating factor. A number ofthese mast cell constituents play a role in the inflammatory responsefunctioning as chemoattractants, activators and spasmogens.Additionally, a number of these constituents are activated and releasedin response to neural stimulation and play a role in neural sensoryadaptation systems. Histamine is well known to produce itching sensationcausing a compulsion to scratch or stimulate the activated area.Histamine also causes pain in patients with genetic predisposition todevelop essential headaches.

An especially important cytokine identified as being important toinflammation and pain is tumor necrosis factor alpha. Tumor necrosisfactor alpha has been identified in activated mast cells, and plays arole in modulation of mast cell activity. (Cocchiara et al. (1999)Histamine and Tumor Necrosis Factor-alpha Production from Purified RatBrain Mast Cells Mediated by Substance P. Neuroreport 10(3):575-8;Olejnik et al. (1998) Tumor Necrosis Factor Alpha (TNF-alpha) ModulatesRat Mast Cell Reactivity. Immunol. Lett. (2-3): 167-71). Anti-tumornecrosis factor, as well as other pre-formed and newly formed mediatorsare autocoids which are reduced when suppressing mast-cell releases.

The botulinum toxin formulations of the instant invention are given in atherapeutically effective dose to reduce inflammation, and may be usedin any application in which inflammation is present or to augment otherinflammatory agents. The administration may be by injection, topicalapplication, or other means to assure a therapeutically effective dosedelivered to the site. Not only is the subject treatment efficacious indisease treatment normally associated with the occurrence ofinflammation, it is also efficacious in the treatment of other diseases.Note that mechanical or adjuvant chemical activity may be necessary toincrease penetration by topical application.

Urticaria refers to the formation of hives occurring usually in responseto allergic reactions to pollens, foods, dander or other forms ofantigens. The process often involves binding of allergens to the IgEreceptor of the mast cell membrane bound IgE, causing release ofpreformed mediators such as histamine and serotonin as well as newlyformed mediators from arachadonic acid such as prostaglandins andleukotrienes, platelet activating factor, kinoginase and tryptase, aswell as cytokines. A late response can be seen after an allergicurticaria reaction which may be painful.

Urticaria may be provoked by non-allergens, including codeine, morphine,compound 48/80, synthetic ACTH, and anaphylatoxins C3a, C5a. Important,relative to the case observation, is the reactivity of mast cells toacetylcholine. (Fantozzi et al. (1978) Release of Histamine from RatMass Cells by Acetycholine. Nature 273 (5662): 473-4).

Mast cells are known to be abundant around blood vessels in the scalp,orbit and lids, and are thought to be important in allergicconjunctivitis. (Allensmith et al. (1981) Percentage of DegranulatedMast Cells in Vernal and Giant Papillary Conjunctivitis. Am. J.Ophthalmol. 9: 71-75; Henriquez et al. (1981) Mast Cell Ultrastructure,Comparison in Contact Lens-associates Giant Papillary Conjunctivitis andVernal Conjunctivitis. Arch. Ophthalmol. 99: 1266-1272). Mast cellreactivity has been associated with hayfever blepharoconjunctivitis,asthma, allergic rhinitis, and allergic forms of eczema. Mast cells arealso seen abundantly in inflammatory responses in rheumatoid arthritisand inflammatory bowel disease.

Mast cells are closely associated with Type-1 hypersensitivityreactions. In such reactions, the typical response involvessensitization with an antigen, formation of immunoglobulin, IgE class,binding of immunoglobulin to the external cell membrane by its FcEreceptor, and setting the stage for hypersensitivity to the secondexposure to the antigen. Upon second exposure, IgE reacts with theantigen effect in a degranulation response of the mast cell, in whichthere is a release of preformed mediators such as histamine andserotonin, platelet activating factor, and newly formed mediators suchas leukotrienes, prostaglandins, tryptase, kininogenase which effectvasodilatation, vascular permeability, micro thrombi, edema, mucoussecretion. The response persists manifesting a late response after 8hours. The late response is associated with pain as described by Roit,I., Brostoff, J., Male, D., Immunology 5^(th) Edition Mosby, 1998.

Internal inflammatory diseases may also be treated with botulinum toxin.In the past, it was thought that the tissue mechanisms associated withusing chemodenervating agents have solely involved the use of botulinumtoxin as a means of causing muscle relaxation or to produce certainautonomic effects blocking decreased sweating. Although there have beenconditions treated by chemodenervating agents which have had associatedinflammatory reaction as a part of the clinical syndrome, the concept ofmuscle relaxation induced by such agents has been thought to be themechanism by which such agents induce the beneficial effects. It has nowbeen found that the subject agent has useful anti-inflammatoryproperties capable of blocking ocular surface allergic inflammation inman and animal models, as well as generalized inflammation within thedenervation field created.

For treatment, the practitioner defines a fixed anatomic area in whichsymptomatic and/or destructive inflammatory processes are occurring.Knowledgeable of dose related diffusion properties and potency of thepreparation being used, the practitioner defines the anatomic area to betreated. Avoiding critical structures, e.g. blood vessels, nerves andanatomic cavities, the practitioner injects a fixed dosage of thechemodenervating agent so as to create a denervation field reducing theintensity of tissue destruction occurring within the area of treatment.Such a field can be defined internally, e.g. stomach mucosae-gastrits,joint-arthritis and muscle myositis. Follow-up involves monitoring forthe cardinal sign of inflammation-pain redness, edema and discharge.Adjuvant therapy with other anti-inflammatory agents would becontemplated.

One of the most devastating chronic internal inflammatory diseases isrheumatoid arthritis, characterized by joint and periocular involvementand chronic inflammatory causing destruction of cartilage andligamentous structures involving joints throughout the body. Immunologiccauses have been cited as the underlying pathologic mechanism of thechronic destructive process, and mast cells have been noted in largequantities within the tissue pannus surrounding joints afflicted. Edema,joint effusions, stiffness, spasms, pain, and erythema, are allcomponents of the arthritis involved regions. Multiple anti-inflammatoryagents have been tried, with variable results to suppress thedestructive effects of this systemic disease on bone and joints.

The formulations of the instant invention offer a means of localizedapplication of an anti-inflammatory agent which is injected directlyinto joints or perarticular muscular tissues which creates an effect onthe rapid inflammatory response and peripheral neural elements governingthe inflammatory response. The application may be repeated at 3-monthintervals and at titrated doses by clinical methods so as to limit anyweakness within the injected region.

J. COSMETIC APPLICATIONS

Lines and wrinkles of the skin are the products of multiple causes thatreduce the collagen and fat content of the skin, including aging and sunexposure. Aging produces wrinkles that may be characterized as finelines that disappear when the skin is stretched. Wrinkles and linesresulting from sun damage are coarser and deeper and do not disappearwhen the skin is stretched. The treatment for wrinkles varies with thedegree of severity.

In some cosmetic applications, the botulinum toxin formulations of thepresent invention may be administered to the muscles of the face,including the forehead and eye area, to reduce lines and wrinkles. Thedisclosed botulinum toxin formulations may be administered through avariety of modalities including surface application, subcutaneous andintramuscular injection. Specifically, botulinum toxin may be used, forexample, to treat glabellar frown lines, crow's feet, horizontalforehead lines, nasolabial fold, mental crease, upper lip, platysmalbands, horizontal neck lines and wrinkles of the lower part of the face.Generally, one to five injections are given per muscle. The selection ofmuscles and the number of injections per muscle, however, depend on thedesired effect and the severity of the lines and wrinkles and are withinthe skill of the treating physician. Administration of botulinum toxinproduces smoothing of the skin and reduction of fine lines andsuperficial wrinkles in the area of treatment.

The botulinum toxin formulations of the present invention areparticularly suitable for use in methods for cosmetically modifyingsoft-tissue features. In particular, these soft tissue features arefeatures of the face and neck. For example, the disclosed formulationsmay be used to alter the shape and volume of facial features such as thelips. Hypervolemic lips, for example, are anatomically caused by one ormore of the following structural deviations: 1) excessive tone of lipretractor function of the certain facial muscles such as levator labiisuperioris, zygomaticus major and minor, levator labii inferioris,platysma, and depressor labii inferioris; excessive prominence anddevelopment of orbicularis oris muscle; and excessive non-muscular softtissue volume within the lip itself. As a consequence of the longduration of botulinum-toxin-induced neuromuscular blockade, catabolismoccurs within the innervated, striated muscle that produces shrinkage ofmuscle fiber and decreased muscle bulk and size. Consequently,administration of the disclosed formulations to muscles of the lipprovide a method to reduce the shape and volume of the lips. Theformulations disclosed herein, may be injected one or more locations andmuscles to produce cosmetic modification of the soft tissue in the areaof administration. Multiple administration of botulinum toxin may berequired to achieve the desired degree of muscle shrinkage and thecosmetic modification of soft tissues.

K. CUTANEOUS DISORDERS

The botulinum toxin formulations of the present invention may also beused to treat a variety of cutaneous disorders, including hypersecretiondisorders of the meibomian glands (chalazion), sebaceous glands(hordeola) and sweat glands (hyperhydrosis). Chalazion is a chronicgranulomatous enlargement of a meibomian gland of the eyelid. Thisdisorder is characterized by hypersecretion of meibum from the meibomianglands. This hypersecretion leads to an accumulation of fatty materialsthat that form lesions that occlude the ductal elements of the gland,leading to an encroachment of the occlusion into the surrounding tissue,which further induces an inflammatory response. Similarly, hordeola ischaracterized by hypersecretion of sebum from sebaceous glands.Individuals suffering from Chalazia and/or hordeola are often treated bywarm compresses or lid soaps which mechanically remove the excesssecretion. This approach is often ineffective. The use of antibacterialeyedrops are occasionally effective, but rarely cure the underlyingproblem—hypersecretion of the meibomian and sebaceous glands that causesinflammation. Patients usually undergo multiple surgical procedures toremove fatty secretions and associated inflammatory cells within theglands to effect relief. Such procedures are painful and occasionallyresult in lid scarring and misdirection of the eyelashes. The presentinvention, however, provides an improved method of treating subjectssuffering from Chalazion, hordeola and cutaneous infections, comprisingthe administration of botulinum toxin to reduce or prevent the secretionof meibum and sebum from meibomian and sebaceous glands, respectively.

Chalazia occurs as a chronic deep inflammation of the lid associatedwith the accumulation of lipid material within macrophages (epithelioidcells) surrounding meibomian glands within the tarsal plate of theeyelid. The inflammation is characterized as a granulomatous-typeinflammation associated with lipid and cellular lesions within softtissues. In the case of chalazia, the lesions are formed by thesecretion of the meibomian glands, the glands which contribute to theouter layers of the tear film covering the ocular surface. Histologicalanalysis of these lesions reveal clear regions representing the lipidmaterial, surrounded by polymorphonuclear leukocytes, plasma cells,giant cells, and lymphocytes.

Hordeola presents a similar pathologic process, however, these lesionsoccur from occluded sebaceous glands at the extreme of the eyelidmargin. The resulting occlusion and excess sebum produces aninflammatory reaction similar to that observed in chalazion.

Chalazion formation has been associated with hypersecretion of thelipid-rich meibum from the meibomian gland. Alterations in the lipidcomposition of meibomian secretions, including free fatty acid andcholesterol content, have also been linked to Chalazion, producing tearfilm instability, irritation of conjunctival and corneal epithelium, andincreased susceptibility to bacterial and fungal infections. Althoughnumerous organism have been identified in the infections frequentlyassociated with Chalazion, the most common isolated bacteria fromblepharitic eyelids include species of Staphylococcus, Corynebacterium,and Proprionibacterium. Staphylococcus aureus has been thought toflourish on hypersecretion of meibomian and related eyelid glands. Insummary, the pathophysiology of chalazia and hordeola involves: 1)altered meibomian secretion and hypersecretion; 2) inflammation fromsecretion backup into soft tissue of the lid; and 3) secondaryinflammation.

The methods of the present invention may also be used to treat pathologyassociated with the occlusion of sebaceous gland ducts, and theresultant inflammation and infection in areas other than the eyelid(e.g. folliculitis).

The present invention also provides methods of treating a bacterial orfungal cutaneous infection in a subject in need thereof, comprising thestep of administering a therapeutically effective amount of acomposition comprising botulinum toxin to the subject, wherein thecomposition reduces cutaneous bacterial or fungal growth. In oneembodiment, the infection is caused by an organism selected from thegroup consisting of: Staphylococcus; Streptococcus and Moraxella.Preferably, the methods of the present invention treat bacterial offungal cutaneous infections in the eyelid, scalp, feet, groin, andarmpit.

The present invention also provides methods of reducing a sebaceous ormucous secretion on a body surface in a subject in need thereof,comprising the step of administering a therapeutically effective amountof a composition comprising botulinum toxin to the subject, wherein thecomposition reduces sebaceous or mucous secretion.

EXAMPLES

The following Examples serve to further illustrate the present inventionand are not to be construed as limiting its scope in any way.

Example 1 Treatment of Blepharospasm

The subject is a 52-year-old female with severe bilateral involuntaryblepharospasm. Involuntary movements have prevented her from driving andmaintaining gainful employment. BOTOX® was administered by injection onfive separate occasions without producing any significant clinicalimprovement. Surgery was performed to remove a portion of theprotractors of eyelid closure (orbicularis oculii). No lastingimprovement was observed.

The albumin content of the BOTOX® was altered by adding 5,000 μg humanserum albumin to a vial of BOTOX® (100 LD₅₀ units). The resultingcomposition has an albumin concentration of 2,750 μg/cc (0.018 LD₅₀/μgalbumin). Administration of 60 LD₅₀ units of the high-albuminpreparation produced a nearly complete resolution of symptoms. Thehigh-albumin concentration was clinically effective even when used insubsequent administrations (4 injection cycles) for over two years.

Example 2 Treatment of Hemifacial Spasm

The subject is a 62-year-old male with a history of bilateral hemifacialspasm. Botulinum-toxin therapy using BOTOX® had been ineffective. Thespasms impaired his day to day ability to function. Decompression of afacial nerve was attempted surgically on two separate occasions. Bothsurgeries proved ineffective in attaining acceptable relief ofinvoluntary facial spasms and produced deafness in one ear.

The albumin content of the BOTOX® was increased by adding human serumalbumin sufficient to achieve a concentration of 5,250 μg/cc (0.00952LD₅₀/μg albumin). Administration of 30 LD₅₀ units of the high-albuminpreparation proved highly effective and substantially relieved theclinical symptoms.

Example 3 Treatment of Hemifacial Spasm

The subject is a 66-year-old man with right hemifacial spasm. Althoughhe was successfully treated with BOTOX® for 11 years, resistancedeveloped that rendered further injections ineffective.Immunologic-resistance testing, using a remote point injection,demonstrated an absence of circulating antibody. A trial of anotherbotulinum toxin formulation, MYOBLOC®, was also ineffective at relievingsigns and symptoms.

The albumin content of BOTOX® was increased by adding human serumalbumin sufficient to achieve a concentration of 5,250 μg/cc (0.00952LD₅₀/μg albumin). Administration of 40 LD₅₀ units of the high-albuminpreparation proved highly effective and substantially relieved theclinical symptoms.

Example 4 Treatment of Benign Essential Blepharospasm

The subject is a 72-year-old university president who was diagnosed withbenign essential blepharospasm. Four prior injections of the standardBOTOX® preparation failed to achieve any significant improvement. Thesubject was referred for possible surgical removal of muscle and nerveto weaken muscles necessary for eyelid closure. Instead, a high-albuminpreparation of botulinum toxin was administered to the usual injectionssites that are specific for benign essential blepharospasm. Thehigh-albumin preparation was produced by adding 12,250 μg/cc (0.004LD₅₀/μg albumin). Administration of 60 LD₅₀ units of the high-albuminpreparation achieved excellent results when the administration of theconventional BOTOX® formulation had failed. Three months after theinitial administration of the high-albumin botulinum toxin preparation,40 LD₅₀ units of a high-albumin preparation comprising 25,000 μg albuminper 100 LD₅₀ units (0.002 LD₅₀/μg albumin) were administered andproduced greater than 80% relief of the clinical symptoms ofblepharospasm.

Example 5 Treatment of Blepharospasm

The subject is a 67-year-old female with blepharospasm that was notresponsive to BOTOX® injections. Surgical removal of nerve and musclefailed to provide any relief from involuntary eyelid closures.

Albumin was added to a conventional BOTOX® preparation to produce ahigh-albumin preparation of botulinum toxin with a concentration of50,250 μg albumin/cc (0.001 LD₅₀/μg albumin). Injection of 50 units thehigh-albumin preparation produced a greater than 50% reduction ofsymptoms.

Example 6 Treatment of Blepharospasm

The subject is a 77-year-old male who noted tachyphylaxis followingrepeated botulinum toxin injections. Conventional formulations ofbotulinum toxin type B were injected without relief of blepharospasm.

Human serum albumin and 0.5 cc Healon® (hyaluronate) were both added toa 100 LD₅₀ units of botulinum toxin type A)(BOTOX®). The high-albuminpreparation produced contained 25,500 μg albumin per 100 LD₅₀ units(0.005 LD₅₀/μg albumin). Administration of 60 LD₅₀ units reduced theclinically-observed involuntary-eyelid contractions.

Example 7 Treatment of Essential Blepharospasm

The subject was a 66-year-old female with essential blepharospasm.Repeated treatment with BOTOX® (type A), using a range between 40 to 300LD₅₀ units, produced no therapeutic benefit. Botulinum toxin type B(MYOBLOC®) was administered at a dose of 10,000 LD₅₀ units within theperiocular region and also failed to produce any relief.Bilateral-facial neurectomy also failed to produce any substantialrelief of symptoms. Additional surgical procedures to remove musclesnecessary for eyelid closure were similarly ineffective.

Human serum albumin was added to a 100 LD₅₀ units of botulinum toxintype A (BOTOX®). The high-albumin preparation produced contained 12,750μg albumin per 100 LD₅₀ units (0.00196 LD₅₀/μg albumin). Administrationof 50 LD₅₀ units produced substantial relief of symptoms for a period ofthree to four months, when other formulations and surgical approacheshad failed.

Example 8 Treatment of Severe Chronic Blepharospasm

The subject is an 83-year-old male with severe chronic blepharospasm.The subject had developed ptosis, a diffusion side effect, afterrepeated treatments with therapeutic doses of conventional botulinumtoxin formulations. The emergence of ptosis complicated the treatment ofthis subject by requiring lower doses of botulinum toxin. The lowerdosing proved less effective.

The patient received an a high-albumin formulation of botulinum toxinthat was produced by mixing 25,000 μg human serum albumin 100 LD₅₀ unitsof BOTOX®. The high-albumin preparation contained 12,750 μg albumin percc (0.004 LD₅₀/μg albumin). Using the high-albumin preparation, 60-70LD₅₀ units were administered with excellent clinical results and noevidence of ptosis after the therapy. The enhanced sequestration of muchhigher concentrations of botulinum toxin depressed the spread of theneurotoxin into the muscles within the eye socket.

Example 9 Treatment of Essential Blepharospasm

The subject is a 67-year-old woman with essential blepharospasm. Thesubject underwent treatment with conventional formulations of botulinumtoxin without relief. In addition, these treatments produced ptosis.

A high-albumin botulinum toxin composition (20,000 μg albumin per cc;0.0025 LD₅₀ BOTOX®/μg albumin) was administered to the subject with aresultant clinical improvement of the blepharospasm and nodiffusion-related side effects (ptosis).

TABLE 4 Comparison of albumin concentrations used in Examples 1-9 withother formulations. High- Albumin Albumin Preparation BOTOX ® DYSPORT ®MYOBLOC ® Concentration (LD₅₀/μg (LD₅₀/μg (LD₅₀/μg (LD₅₀/μg Example(μg/cc) albumin/cc) albumin/cc) albumin/cc) albumin/cc) 1 2,750 0.01800.2 5 10 2 5,250 0.0095 0.2 5 10 3 5,250 0.0095 0.2 5 10 4 12,500 0.00400.2 5 10 25,000 0.0020 5 50,250 0.0001 0.2 5 10 6* 10,200 0.0050 0.2 510 7 25,000 0.0020 0.2 5 10 8 12,500 0.0040 0.2 5 10 9 20,000 0.0025 0.25 10 LD₅₀/mcg albumin/cc for BOTOX ®, DYSPORT ®, MYOBLOC ® given fordirect comparison

Example 10 Preparation of a High-Albumin Composition of Botulinum Toxin

After quantitating the biologic effect by dilution of purified botulinumtoxin, a quantity of albumin is added to the lyophilized or liquidmaterial in a quantity sufficient to exceed 500 mg per 100 LD₅₀. Theincreased albumin binds to botulinum toxin and enhances sequestration ofthe injected neurotoxin providing for better saturation of neurotoxinreceptors and improved clinical effect.

Example 11 Preparation of a High-Albumin Composition of Botulinum ToxinFurther Comprising Hyaluronate

After quantitating the biologic effect by dilution of purified botulinumtoxin, a quantity of albumin is added to the lyophilized material in aquantity sufficient to exceed 500 μg per 100 LD₅₀ units. Additionally,another sequestration agent, which further enhances sequestration, isadded to keep the botulinum neurotoxin from diffusing away from theinjections site. Such a sequestration agent includes but is not limitedto a diluted solution of sodium hyaluronate. The increased albuminnon-covalently binds to botulinum toxin and an enhances thesequestration of the neurotoxin providing better saturation ofneurotoxin receptors and, consequently, an improved clinical effect.

Example 12 Preparation of a High-Albumin Composition of Botulinum ToxinFurther Comprising Collagen

After quantifying the denervating effect of a botulinum neurotoxin bydilution of a purified botulinum toxin, albumin is mixed with thelyophilized botulinum neurotoxin in a quantity sufficient to exceed 500μg albumin per 100 LD₅₀ units. Additionally, another physical agent,which further enhances sequestration, is added to keep botulinumneurotoxin from diffusing away from the injections field. Such an agentwould be a diluted mixture of animal or human collagen. The increasedalbumin non-covalently binds botulinum toxin, an enhances sequestrationof the neurotoxin, providing better neurotoxin receptor saturation andimproved clinical effect.

Example 13 Preparation of a High-Albumin Composition of Botulinum ToxinComprising a Recombinantly-Produced Botulinum Toxin-Albumin FusionProtein

Botulinum toxin is produced as a fusion protein with albumin therebyproducing an albumin molecule that is covalently linked to a botulinumtoxin. The fusion protein is tested using the mouse LD₅₀ bioassay todetermine the effective amount. The regional denervation rabbit ptosisbioassay and mouse hindlimb bioassay may be used to confirm theeffective amount of a composition comprising the fusion protein. Aclinical-dose-escalation study would be further used to confirm andrefine effective amount.

Example 14 Resistance to Botulinum Toxin after Treatment for CosmeticIndications

A 44-year-old woman received multiple injections of botulinum type Atoxin for the effacement of glabellar rhytides and crowsfeet. After 8injections, she noted the medication no longer was effective. A maximalfacial dose of 80 U failed to produce any significant benefit.Substitution of botulinum toxin type B caused effacement indicatingresistance to botulinum type A.

Example 15 Preparation of a High Specific Activity Botulinum ToxinPreparation with Increased Potency Per LD₅₀ Unit for the Treatment ofFacial Rhytides

Botulinum toxin formulated as pure neurotoxin (accessory proteinsremoved and high specific activity of about 80 LD₅₀ units per nanogram),1 mg trehalose, with 4000 mcg of albumin per 100 LD₅₀ was used to treatglabellar rhytides injected over 5 sites between and over the eye-browregion in an FDA-approved phase-1-human-clinical trial. The purpose ofthe study was to determine safety and efficacy using a dose-escalationparadigm. An initial dose of 6.25 U or 1.25 U per injection site (5) wasselected. This starting dose (6.25 U per injection cycle) was selected,because it was expected to be below a therapeutically effective dose.The initial dose was chosen by a panel of expert physicians, and wasaccepted by regulators at the United States Food and Drug Administrationas below that necessary to produce any therapeutic effect. Surprisingly,this low dose proved efficacious in about 75-80% of patients, a resultunexpected both in view of experience with pre-clinical animal modelsand prior clinical experience with BOTOX®. The response rates werecorroborated by multiple primary endpoints, using both patient andphysician assessment. Furthermore, the unexpected favorable responselasted 10 weeks in over 50% of the patients in this dosing cohort, aresult similar to a 20 U dose of BOTOX®.

TABLE 5 Effect of 6.25 Units of High-Potency Botulinum Toxin Formulationon Treatment of Glabellar Rhytides. A. Investigator Baseline Δ ResponseSubject Rest Frown Day 22 (CFB #steps) Day 29 Δ Response Day 71 ΔResponse 6.25 U (R) (F) R F R F R F R F R F R F 1101 1 2 1 2 0 0 1 2 0 01 2 0 0 1102 1 2 1 1 0 1 1 1 0 1 1 1 0 1 1103 2 3 2 2 0 1 2 2 0 1 2 2 01 1104 0 2 0 1 0 1 0 1 0 1 0 1 0 1 1105 1 3 1 2 0 1 1 2 0 1 1 2 0 1 11061 3 1 2 0 1 2 2 −1 1 2 2 −1 1 1107 2 2 2 2 0 0 2 2 0 0 2 2 0 0 1108 2 32 2 0 1 2 2 0 1 2 2 0 1 % Primary Endpoint Responders = 75 75 75 B. SGAΔ Response Subject Baseline Day 22 (% CFB) Day 29 Δ Response Day 71 ΔResponse 6.25 R F R F R F R F R F R F R F 1101 69 93 19 32 72.5 65.6 831 88.4 66.7 29 48 57.9 48.4 1102 68 82 3 7 95.6 91.5 16 19 76.5 76.8 1728 75 65.9 1103 69 89 59 67 14.5 24.7 63 67 8.7 24.7 69 76 0 14.6 110445 68 2 2 95.6 97.1 1 2 97.8 97.1 8 25 82.2 63.2 1105 55 82 3 25 94.569.5 1 19 98.2 76.8 22 38 60 53.7 1106 85 92 56 66 34.1 28.3 69 69 18.825.0 77 75 9.4 18.5 1107 92 98 72 87 21.7 11.2 73 88 20.7 10.2 73 9320.6 5.1 1108 74 80 28 11 62.2 86.3 22 10 70.3 87.5 58 76 21.6 5.0 %Primary Endpoint Responders = 62.5 62.5 37.5

Table 5 depicts the data obtained from the administration of thehigh-albumin formulation of botulinum neurotoxin type A to eightpatients for the treatment of glabellar rhytides, using an FDA-approvedhuman studies protocol to determine safety and efficacy. The response totreatment was measured using a visual, patient-based self-gradingassessment (SGA) using an analog scale (0-100 mm) and investigator-basedphoto-scale grading (FDA-approved primary endpoints for Phase-1protocol). The visual SGA scale ranges from 0-100 mm. A grading of “0”represents no self-perceived rhytides, whereas a grading of “100”represents a self-perception of extreme or severe rhytides. Grading wasdone with facial muscles at rest and during a forced frown. Thephysician photo-scale ranges from 0-3. A grading of “0” indicates noobservable rhytides and a grading of “3” is indicative of severerhytides with respect to depth, extent, and prominence with facialmuscles at rest and during forced frown.

A 75% response rate for in patients receiving 6.125 Units was obtainedat Day 22 (three weeks) using the physician photo-scale assessment(Table 5A). A 62.5% response rate was to observed by SGA (Table 5B). Asignificant number of the 6.25 U patients sustained the beneficialeffect through at least Day 71 (10 weeks).

The results obtained from this dose escalation study are contrary toexpected outcomes based on prior experience with immunotype A botulinumneurotoxins formulated with 500 mcg or less human serum albumin per 100LD₅₀ U (such as BOTOX®). The package insert for BOTOX®, representing theresults from multiple clinical studies indicates 20 units (4 U perinjection site) as the dose sufficient to diminish glabellar rhytides.

Example 16 Treatment of Rhytides Using a High Specific NeurotoxicityBotulinum Toxin Formulation

Eight patients interested in having glabellar rhytides effaced wereinjected with a composition comprising a botulinum toxin with a specificactivity of about 80 u/ng, 1 mg trehalose and 4,000 mcg human serumalbumin per 100 LD₅₀ Units neurotoxin. Patients received 6.125 U totalin 5 injection sites (1.25 U per site). Patients were followed for 3, 4and 10 weeks for safety and efficacy using a visual analog scale forpatient global self-assessment and a photo-scale rating severity ofrhytides. Surprisingly, the composition described herein produced asubstantial improvement within 72 hours of injection with 75% of casesimproved as assessed using a physicians grading scale and over 62%improvement using a patient self assessment scale (p. 0.01, compared tosaline controls). To achieve similar results, at least 20 U of BOTOX® isnecessary.

Example 17 Determining the Immunogenicity of Botulinum ToxinFormulations

A rabbit model has been used to assess the immunogenicity of varioustoxin preparations. In that model, albino rabbits receive repetitivesublethal injections of various toxin preparations over a period oftime. Rabbit serum is assessed for neutralizing botulinum toxinantibodies by exposing a botulinum toxin to serum from the immunizedrabbit and comparing the denervating activity (denervation potency) ofequivalent dosages of botulinum toxin exposed to the rabbit serumagainst unexposed botulinum toxin. Animal models for muscle denervationare disclosed and described in U.S. Pat. No. 5,298,019, which isincorporated herein by reference in its entirety. An observation ofdecreased denervation potency, when comparing exposed botulinum toxin tothe unexposed toxin, indicates that the toxin preparation inducedneutralizing antibody formation. Similarly, differences in denervationpotency between botulinum toxin preparations indicate differentimmunogenicity of the preparations. Reduced denervation potency isproportional to the immunogenicity of the preparation.

Example 18 Denervation Potency in Rabbit Muscle

In animal models, the potency of a botulinum toxin preparation may bedetermined by measuring the extent of denervation produced when apreparation is administered to a muscle. The longissimus dorsi muscle ofNew Zealand white rabbits are injected with botulinum toxin. Five weeksafter administration of the botulinum toxin, the animals were sacrificedand denervation of the muscle a 0, 15, 30 and 45 mm from the injectionsite was determined by examining muscle-fiber variability (FIG. 1) andacetylcholinesterase activity histochemical staining (FIG. 2).

The longissimus dorsi muscle of twenty-eight (28) white New Zealandrabbits were injected with 5 LD₅₀ Units of a commercial preparation ofBOTOX®. BOTOX® is a composition comprising botulinum toxin type A and500 μg human serum albumin (HSA) per 100 LD₅₀ Units. Thirty-two (32)white New Zealand rabbits were injected with 5 LD₅₀ Units of acomposition comprising botulinum toxin type A and 4,000 mg HSA per 100LD₅₀ Units. Each toxin preparation was reconstituted with physiologicalsaline. The point of injection was marked with a tattoo. The injectionswere made 5 to 8 mm deep directly into the muscle. A control animal wasinjected with the saline diluent. After five weeks, the animals weresacrificed, and muscle biopsies were taken 0, 15, 30, and 45 mm from theinjection site transverse to the spine and in a direction parallel tothe spine on the contralateral side. Tissues were analyzed fordenervation based on muscle fiber variability using standardhematoxylin-eosin staining and acetylcholinesterase staining. Thereversible neurogenic muscle fiber atrophy begins within about 10 to 14days following injection. The general reduction in fiber diameter isoften accompanied by a large degree of fiber size variability. Thespreading of acetylcholinesterase staining (normally localized almostexclusively to the neuromuscular junction) over the sarcolemma of musclefibers occurs over a 3 to 4 week period following injection and isuseful for assessing the denervation activity of botulinum toxinpreparations.

Biopsies were studied microscopically to compare muscle fibervariability with muscle fibers from a saline-injected control animal.Assessment of botulinum toxin activity within muscle was accomplishedusing a four point histologic grading scale (0=normal fiber variability;1=low fiber variability; 3=moderate fiber variability; and 4=extensivefiber variability) based on the severity of muscle fiber neurogenicatrophy and extra-junctional acetylcholinesterase staining intensity.The results are depicted in FIG. 1.

The longissimus dorsi muscle of thirty-two (32) white New Zealandrabbits were injected with 5 LD₅₀ Units of a commercial preparation ofBOTOX®. Nineteen (19) white New Zealand rabbits were injected with 5LD₅₀ Units of a composition comprising botulinum toxin type A and 4,000μg HSA per 100 LD₅₀ Units. Each toxin preparation was reconstituted withphysiological saline. The point of injection was marked with a tattoo.The injections were made 5 to 8 mm deep directly into the muscle. Acontrol animal was injected with the saline diluent. After five weeks,the animals were sacrificed, and muscle biopsies were taken 0, 15, 30,and 45 mm from the injection site transverse to the spine and in adirection parallel to the spine on the contralateral side. Tissues wereanalyzed for denervation based on acetylcholinesterase activitystaining. Assessment of botulinum toxin activity within muscle wasaccomplished using a four point grading scale of acetylcholinesteraseactivity staining (0=no staining; 1=low intensity; 3=moderate intensity;and 4=high intensity). The results are depicted in FIG. 2.

We claim:
 1. A method of treating muscle spasticity in a human,comprising the step of administering by injecting into a muscle of saidhuman patient a dose between 20 to 2250 picograms of botulinum toxintype A per injection cycle, from a pharmaceutical formulation comprisingbotulinum toxin type A and zinc-saturated human serum albumin in anamount greater than 500 micrograms human serum albumin per 100 LD₅₀Units botulinum toxin type A, wherein the administration of saidformulation produces a therapeutic muscle weakness in said humanpatient, and wherein said formulation has specific activity between 100and 250 LD₅₀ Units per nanogram botulinum toxin type A.
 2. The method ofclaim 1, wherein said muscle is the flexor digitorum profundus muscle,and said dose is between 20 to 450 picograms of botulinum toxin type Aper injection cycle.
 3. The method of claim 1, wherein said muscle isthe flexor digitorum sublimus muscle, and said dose is between 20 to 450picograms of botulinum toxin type A per injection cycle.
 4. The methodof claim 1, wherein said muscle is the flexor carpii ulnaris muscle, andsaid dose is between 20 to 450 picograms of botulinum toxin type A perinjection cycle.
 5. The method of claim 1, wherein said muscle is theflexor carpii radialis muscle, and said dose is between 35 to 725picograms of botulinum toxin type A per injection cycle.
 6. The methodof claim 1, wherein said muscle is the biceps brachii muscle, and saiddose is between 100 to 2250 picograms of botulinum toxin type A perinjection cycle.
 7. The method of claim 1, wherein said zinc-saturatedhuman serum albumin enhances an endopeptidase activity of said botulinumtoxin type A.
 8. The method of claim 1, wherein said zinc-saturatedhuman serum albumin enhances a denervating effect of said botulinumtoxin type A.